Messi capped a virtuoso individual display with an injury time winner at the Santiago Bernabeu on Sunday, moving Luis Enrique's men back to the top of the table on goal difference.
Madrid will hope to quickly close up a three-point deficit when they face Deportivo La Coruna later on Wednesday, after which they will still have a game in hand on their rivals.
Messi also reached 500 goals for Barcelona with his Clasico brace and a huge banner at Camp Nou honoured his achievement before kick-off.
He made it 501 in the 12th minute and scored a wonderful second after midfielder Andre Gomes netted a double either side of a free-kick from Roberto Torres of Osasuna, who are bottom of the table and on the verge of relegation.
Paco Alcacer got in on the act with a brace of his own after Messi was substituted to a standing ovation and Javier Mascherano contributed to a testimonial feel by netting a first ever Barcelona goal from the penalty spot.
A deep-lying Osasuna gave little away during the opening 10 minutes but, shortly after venturing into the Barca area for the first time, a dreadful error saw them go behind on the break.
Fausto's pass on halfway was slack and Messi did not need a second invitation, intercepting the loose ball, nodding it down and juggling it before lifting a finish over goalkeeper Salvatore Sirigu.
Camp Nou rose in acclaim, with a number of fans replicating Messi's already famous weekend celebration in Madrid by holding their Barcelona shirts aloft.
It remained a patience game for the hosts until their second arrived with half an hour played, after Messi found space 25 yards from goal to see a whipped shot deflected narrowly wide.
The resulting corner was taken short to Ivan Rakitic, whose bouncing cross was greeted by an assured right-footed finish from Gomes.
Torres stunned Camp Nou by reducing the arrears three minutes into the second half.
The midfielder's free-kick was nicely struck but questions will be asked of Barcelona goalkeeper Marc-Andre ter Stegen, whose initial movement to his left – behind his defensive wall – left him with no chance.
Sirigu pushed a header from Mascherano on to his post as Barca sought to restore their cushion, but Torres had an equaliser in mind when he fired goalwards and Ter Stegen stood firm.
Any jitters among the home faithful were settled in the 58th minute when Gerard Pique took down Rakitic's left-wing corner on his chest, shot against the base of the post and saw Gomes slot in the rebound.
Following that simple finish, Messi then demonstrated the brilliance he makes look routine by dropping deep to receive possession, scampering towards the edge of the box and caressing a left-footed shot into the corner.
The five-time Ballon d'Or winner then departed to a standing ovation – the pressure well and truly back on a Madrid side who have seen quite enough of him this week.
Barca continued to enjoy themselves in their talisman's absence, with Alcacer sliding to lift a finish over Sirigu after Osasuna failed to deal with Arda's hopeful 65th-minute ball into the box.
Merida clumsily tripped Denis Suarez to give Mascherano the chance to finally become a Barcelona goalscorer in his seventh season with the club. The Argentine clattered the ball high into the net, prompting huge cheers in the stands and hysterics on the home bench.
500 - Javier Mascherano has scored the 500th goal for Barcelona under Luis Enrique in all competitions. Milestone. pic.twitter.com/firbgDjc46— OptaJose (@OptaJose) April 26, 2017
Alcacer then steamed clear to coolly round Sirigu and completed the rout four minutes from time, sending a resounding message to Zinedine Zidane and company.
Key Opta stats:
- Javier Mascherano needed 194 games and 31 shots to score his first goal for Barcelona in LaLiga.
- The Argentine scored the 500th goal for Barcelona under Luis Enrique in all competitions (501 in total).
- Lionel Messi has scored more goals than any other player in Europe's top five leagues this season (33).
- The Argentine has scored 49 goals in 47 competitive games for Barcelona this season.
- Messi has scored a brace in seven of his last 11 LaLiga games.
- Barcelona have won 10 LaLiga games at the Camp Nou for the first time with Luis Enrique as coach.
- Paco Alcacer has scored his first brace for Barcelona in all competitions.
- Ivan Rakitic has assisted in consecutive LaLiga games for Barcelona for the first time (three assists).
- Sergio Busquets attempted 139 passes against Osasuna (129 successfully), the highest for a player in a LaLiga game this season.
- Salvatore Sirigu has conceded seven goals in a league game for the second time, the first one since 2011 against Udinese in Serie A.