The in-form England striker led the line confidently and was rewarded with two fine goals either side of Gareth Bale's first goal of the season as Spurs ran out winners.
A well-worked move on the right flank saw Gylfi Sigurdsson slide Aaron Lennon in behind the home side's defense and his pull back was clinically converted by Defoe from 10 yards. A late tap-in from Bale and a brilliant counterattack saw Defoe double his tally for the afternoon with a fine finish.
The Royals took until the 85th minute to register their first shot on target and a late Hal Robson-Kanu goal proved to be nothing more than a consolation for a side that was outplayed for vast chunks of the fixture.
Villas-Boas handed Mousa Dembele his full debut, replacing Jake Livermore while Kyle Naughton was given his first Premier League start in place of Benoit Assou-Ekotto, who picked up an injury on international duty.
The home team named 22-year-old Alex McCarthy as the replacement for the out-of-form Adam Federici and he was immediately called into action as a confident Tottenham threatened in the early stages.
Bale's cross from a wide free kick was met by Sigurdsson, but McCarthy reacted well to parry the header away from goal and was swift to recover his position to enable him to deflect Jan Vertonghen's near-post shot behind for a corner.
Spurs played with authority and although Bale missed the target with a deft flick from a Vertonghen long-ball, Defoe made no mistake when he caught his first clear sight of goal.
Sigurdsson, who was returning to the club that gave him his chance in English football, showed that he was more than just a goalscoring midfielder, threading a well-weighted pass in between Kaspars Gorkss and Ian Harte for Aaron Lennon to scamper onto.
Instead of driving to the byline, Lennon cut the ball back to Defoe immediately and watched as the in-form England international stroked a first-time shot low and into the far corner to give Spurs a 1-0 lead inside 20 minutes.
Sandwiched between Spurs' peppering of the Reading goal came the home team's best chance of a goal. A deep cross saw Alex Pearce leap with Kyle Walker and although the Tottenham fullback handled the ball, Webb opted to give Villas-Boas' side a free kick for the over-zealous nature of Pearce's challenge. Frankly, it could have gone the other way.
Though the Royals felt aggrieved at this decision, there was little else they could complain about in a half in which Tottenham was markedly the better side.
Defoe's sublime first touch from Naughton's long-ball almost saw him create a second, but his third touch – the shot – was toe-poked wide.
The diminutive striker kept hassling the Reading defense though, and his presence led McCarthy to take a heavy fast touch inside his box. Defoe stole the ball from the shot-stopper, ran wide before squaring to Sigurdsson but watched as Pearce made an heroic goal line block from the Icelander.
Aside from a late Jobi McAnuff 20-yard effort, Reading had very little to show for a first half that was dominated by Tottenham.
The second half, predictably, was a different story – at least in the initial stages. Jem Karacan was replaced by Adam le Fondre at the break as Brian McDermott looked to utilize a more direct approach.
The impressive Vertonghen remained diligent at the back alongside William Gallas, and although Reading roused the home fans for a time, Spurs looked comfortable enough.
Indeed, the renewed purpose of the home side did provide Villas-Boas' team with counterattacking opportunities and one such move sparked by Dembele saw his cross-shot deflect off Defoe and up and over the crossbar.
McDermott's side could not create a legitimate scoring chance and with the attacking talent on offer, Spurs would eventually pounce.
Firstly, great work on the right from Walker saw him brush past two defenders before squaring the ball from the byline for Bale to meet and tap home fortuitously with his right foot.
As Villas-Boas punched the air in celebration, Defoe hounded Gorkss into yielding possession on the halfway line and initiated another threatening counter. This time, with Bale darting to his right, opening up space, Defoe glided past Pearce's left side and slammed the ball back across McCarthy for his second goal of the game.
Late substitutions allowed Tom Huddlestone further minutes on the pitch and gave Clint Dempsey his debut as Villas-Boas looked to close out the game safely.
However, although Gallas cleared Le Fondre and Reading's first shot on target off the line with five minutes to play, the substitute striker showed great invention in the final two minutes to clip a cross over Brad Friedel and provide the assist for the onrushing substitute Hal Robson-Kanu.
For McDermott, he will look back at the game with frustration. Tottenham outplayed his side, but as will be the case this season, Reading needs to find other ways to threaten stronger opposition and although the club found a consolation goal, they will know they must improve at both ends to collect points in the Premier League.
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