Chelsea, under interim manager Roberto di Matteo, stands in the way of defending champion Barcelona and a third Champions League showpiece in four years.
Madrid faces a freescoring Bayern Munich team eager to reach the final to be played at their Allianz Arena on May 19.
"We'll pull everything out, we'll fight, give everything to make it," Bayern attacking midfielder Thomas Mueller said ahead of Tuesday's first leg between teams who have won Europe's biggest club prize 13 times between them.
Barcelona, bidding to become the first team to retain the Champions League in its current format, have been helped along by the imperious scoring exploits of Lionel Messi, whose haul of 14 goals so far has broken the competition for one campaign set by Ruud van Nistelrooy in 2002-03.
The three-time world player of the year's overall tally for the season currently stands at 63 and he will be eyeing more in Wednesday's first leg against a Chelsea defence that has looked rocky at times this season.
The two Spanish giants must juggle their Champions Leagues ambitions with their fight for the domestic title, which will hinge upon the second 'Clasico' of the season in Barcelona in between the two European semifinal legs.
"We are so proud to arrive at this point of the season with so many opportunities, so many challenges for the prizes," Barcelona coach Pep Guardiola said. "Now the mental (part) controls the situation of the stress ... All the players know at the end of the season that if they lose they are finished.
"With this mentality we will travel to London to play Chelsea."
Chelsea's decision to change managers last month, bringing in Di Matteo on a temporary basis in place of the sacked Andre Villas-Boas, has revitalized the team in cup competitions at least.
The Blues thrashed London rivals Tottenham 5-1 on Sunday to reach the FA Cup final but sits sixth in the Premier League and looks unlikely to play in next season's Champions League, unless it can win the competition this year and get a berth as defending champion.
"It's good to keep this positive momentum going," Di Matteo said. "We go into the game with confidence. Winning brings that positivity within the team. It make everybody feel better."
The teams last met at this stage in 2009, when Norwegian referee Henning Ovrebo denied several Chelsea penalty appeals in a 1-1 draw in the second leg, which meant Barcelona advanced on away goals before going on to win the competition.
The specter of "unfinished business" was raised by Chelsea midfielder Frank Lampard after the team's quarterfinal win over Benfica.
Chelsea is likely to be without David Luiz (hamstring) but fellow center back John Terry is ready to play despite revealing he has been struggling with cracked ribs since the Benfica match.
Playing a record fifth straight Champions League semifinal, Barcelona is still without long-term absentee David Villa but Dutch midfielder Ibrahim Afellay is available again after a knee injury.
If Barca will look to Messi for goals, Madrid will turn to Cristiano Ronaldo, who has eight for the competition to add to his 41 in the Spanish league.
Ronaldo and fellow forwards Karim Benzema and Gonzalo Higuain have combined to score 107 goals in all competitions this season.
"We've got enough players up front who can score goals and we want that to continue," Madrid defender Marcelo said. "We have to take advantage of Cristiano's goals, there are no limits to his abilities."
Madrid and Bayern represent Europe's top rivalry with the pair of European powerhouses having met a record 18 times in the knockout stages, with the Germans winning 10 of those series - including the most recent five years ago.
Mourinho won the Champions League with Inter Milan in 2010 by beating Bayern in the final at Madrid's Santiago Bernabeu stadium. The former Chelsea boss also has also won the title with Porto and is now seeking to do so with a third club.
Like Madrid, Bayern has no fresh injury worries and is pinning all its hopes this season on winning a fifth European crown after giving up on the Bundesliga title following a loss to Borussia Dortmund last week.
"Everyone knows what's coming. It's the toughest opponent of the season," Bayern's Toni Kroos said.
The Bavarian club has won 13 of its past 14 Champions League games at home, and is unbeaten in nine games against Madrid in Munich.