Knocking Barca out of Europe's elite club competition after winning the King's Cup final against them last week could signal the start of a power-shift in Spanish football away from Catalunya and back to Madrid.
The Portuguese coach has always managed to rile Barca, where he spent some of his formative years as an assistant to Bobby Robson in the 1990s, and guided Inter Milan to victory against them in the semis last year on the way to Champions League glory.
Mourinho also crossed swords with Barca three times during his spell at Chelsea, twice bettering them over a 'two-match series'.
His ability to ruffle the Catalan team's feathers knows no end and he has outwitted them so far in the unprecedented 'four-match series' between the world's two richest clubs.
A 1-1 league draw, when Real came back to level with 10 men on April 16, was followed by last Wednesday's 1-0 extra-time victory in the King's Cup Final, Mourinho's first trophy as coach of the Madrid side.
The league title is all but wrapped up for Barcelona but the tantalising prospect of a 10th European Cup triumph lies ahead for Real, and a third for Mourinho all with different teams.
Barca defender Gerard Pique refused to be drawn on the hot topics of debate after the first two 'Clasicos' such as the quality of the refereeing, the length of the grass at the Bernabeu and Real's aggressive, 'negative' approach.
"The grass is long but I'm not going to cry about it," the Spanish World Cup winner told daily El Pais of the pitch at Wednesday's Bernabeu venue.
"It's normal for them to take particular care over their defence and that they try to stop our passing game. Mourinho will use everything available to him.
POWERFUL TEAM
"At the end of the day they are a powerful team with very good forwards," added Pique.
Breaking coach Pep Guardiola's record of five consecutive 'Clasico' wins, including a 5-0 demolition of Mourinho's team at the Nou Camp in November, and then winning the King's Cup has dented Barca's aura of invincibility.
Real have proved they can match their rivals with ultra-aggressive tactics that have not been to everyone's liking, with the Madrid club having had a player sent off in the last three meetings between the sides.
The two clubs are the competition's top scorers with 24 goals each but the previous two 'Clasicos' have been tense tussles with few sights of goal.
Madrid have packed the centre of the pitch with three holding players led by centre-back turned midfield-enforcer Pepe, ruthlessly denying Barca's playmakers time and space while looking to break quickly.
Sami Khedira's injury means Lassana Diarra will probably replace him in midfield while Mourinho may opt to play without a recognised number nine again as he did in the King's Cup final.
At the back Ricardo Carvalho is suspended but Real's defensive problems are not as acute as Barca's.
The absence of Eric Abidal, Adriano Correia and Maxwell mean the returning Carles Puyol could be as
