Barcelona beat Real Sociedad 1-0 in captain Andres Iniesta's final game at Nou Camp.
Fernando Torres scored twice in his final game in boyhood club Atletico Madrid's coloures as they drew 2-2 with Eibar.
Cristiano Ronaldo and Gareth Bale's goals were cancelled out as Villareal managed a 2-2 draw against Real Madrid.
Barcelona's Andres Iniesta and Atletico Madrid's Fernando Torres set to play their last games for their boyhood clubs.
Lionel Messi said he had spoken to Neymar about rumours linking him with a return to Spain for La Liga rivals Real Madrid.
Barcelona had last tasted league defeat 13 months ago against Malaga, going 43 games unbeaten, but they failed in the 37th match of this season to tarnish an otherwise flawless domestic campaign.
Barcelona lost 5-4 in a wild game at Levante on Sunday, ruining the champion's chance to become the first team to finish a Spanish league season without a defeat under its current format.
Real Madrid did not need resting forward Cristiano Ronaldo to crush Celta Vigo 6-0 at home in its second-to-last match before the Champions League final against Liverpool.
Real Madrid are third in the standings on 72 points and missed out on the chance to move level with city rivals Atletico, who are second on 75.
Barca are two matches away from becoming first team in La Liga since 1933 to complete a season without losing and first since league was expanded from nine teams.
Sergi Roberto will miss the rest of the Liga season and the first match of the next campaign after being given a four-game ban for striking Real Madrid's Marcelo in last Sunday's tension-filled 2-2 draw at the Nou Camp.
Antoine Griezmann, one of Atletico Madrid's key players, has been heavily linked with a move to the Nou Camp at the end of the season.
Barcelona in contact with Antoine Griezmann's agent amid reports the Atletico Madrid striker is set to join the Spanish champions.
El Clasico had as much focus on the players as it did on the referee with Alejandro Hernandez Hernandez dishing out eight yellow cards.
Barcelona's clash against Real Madrid on Sunday saw a series of controversial decisions from the referee that grabbed the headlines.
Real Madrid coach Zinedine Zidane expressed confidence that Cristiano Ronaldo will play against Liverpool in the Champions League final on May 26.
Barcelona remained unbeaten in La Liga as they drew 2-2 against Real Madrid despite Sergio Roberto being sent off at Camp Nou.
Barcelona vs Real Madrid El Clasico Highlights: Barcelona managed to draw 2-2 against Real Madrid despite being a man down.
Espanyol earned the surprise 2-0 win after scoring the first goals in nine visits by an opposing team at the Wanda Metropolitano Stadium.
Barcelona sealed la Liga crown with a 4-2 win at Deportivo La Coruna last Sunday, meaning pride is all that is at stake when they face Real Madrid.