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 comfortable winners
A well-worked move on the right flank saw Gylfi Sigurdsson slide Aaron Lennon in behind the hosts' defence and his pull-back was clinically converted by Defoe from ten yards, before a 71st minute tap-in from Bale was followed by a brilliant counter-attack that 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 were outplayed for vast chunks of the fixture.
Villas-Boas handed Mousa Dembele his full debut, replacing Jake Livermore in midfield, 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 hosts named 22-year-old Alex McCarthy as the replacement for the out-of-form Adam Federici, who had also struggled for fitness in the lead-up to this fixture, and he was immediately called into action as a confident Tottenham threatened in the early stages.
The visitors 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 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 hosts' best chance of opening their own account. A deep cross saw Alex Pearce leap with Kyle Walker and although the Tottenham full-back 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 were markedly the better side.
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The diminutive striker kept hassling the Reading defence though, and his presence led McCarthy to take a heavy first touch inside his box. Defoe stole the ball from the shot-stopper and ran wide before squaring to Sigurdsson but could only watch on as Pearce made an heroic goal-line block to stop 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 early stages. Jem Karacan was replaced by Adam Le Fondre at the break as Brian McDermott looked to utilise a more direct approach.
McDermott's side, for all their bluster though, could not create a legitimate scoring chance and with the attacking talent on offer, Spurs would eventually pounce.
First, 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 and opening up space, Defoe glided to 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 Robson-Kanu.