Sevilla separate the giants of Spanish football in second, just a point behind Madrid, after twice coming from behind to edge a seven-goal thriller 4-3 at rock-bottom Osasuna.
However, Real Madrid also have a game in hand over their title rivals.
Barca's visit to the Basque Country was only eight minutes in when Busquets had to be carried off with ankle ligament damage after a dangerous challenge from Gonzalo Escalante that went unpunished.
Yet, there was little doubt over the outcome once Denis Suarez, who had replaced Busquets, drilled home his first Barca goal into the far corner from outside the box on the half hour mark.
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Neymar missed a one-on-one and Luis Suarez hit the post as Barca missed a series of chances to kill the game before half-time.
However, Messi doubled their lead just six minutes into the second-half when he swept home Luis Suarez's cross for his 28th goal in 26 games this season.
Neymar hadn't scored from open play since October, but brought that run to an end by slotting home Aleix Vidal's low cross in stoppage time.
Record-breaking Sevilla
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Sevilla scored twice in the final five minutes to end Real Madrid's 40-game unbeaten run in all competitions last weekend and have now won five of the seven La Liga games when they have conceded first this season.
"The team did some good things and made mistakes, but from start to finish looked to win the game," said Sevilla boss Jorge Sampaoli.
Osasuna were infuriated Franco Vazquez's vital third goal for Sevilla was allowed to stand despite a push on Oriol Riera 10 minutes from time.
Pablo Sarabia's fine left-footed shot from outside the area sealed the points, but there was still time for Osasuna to pull another goal back when Kenan Kodro headed in from close range.
Atletico Madrid lost further ground on the top three as they needed a spectacular Antoine Griezmann equaliser 10 minutes from time to secure a 2-2 draw at Athletic Bilbao.
The visitors got off to the perfect start when Koke's cross towards Griezmann evaded everyone and flew into the far corner after just three minutes.
However, Atletico were punished for trying to sit on their lead when Inigo Lekue smashed home an equaliser three minutes before half-time.
Athletic then went in front as former Atletico favourite Raul Garcia crossed for Oscar de Marcos to head home.
Griezmann had already had one spectacular finish ruled out by the offside flag, but the French international continued his fine form with a rasping low drive from 25 yards to at least salvage a point for Atletico.