The midfield pair - who have formed part of Carlo Ancelotti's first-choice engine room trident alongside Luka Modric this season - have both been suffering from illness in recent days.
Kroos has sat out Real's wins over Elche and Osasuna in La Liga due to gastroenteritis, while Tchouameni was a last-minute absentee from the weekend's win over the latter after suffering similar symptoms.
Ancelotti initially expressed hope over Kroos's availability for the contest, with the Germany international having had ample time to recover from his viral condition, but Tchouameni was considered more of a doubt.
Neither player was able to train with the team on Sunday, though, and they have both been left out of the Real Madrid squad in a major double blow to the reigning champions.
Left-back Ferland Mendy is also absent as he recovers from a thigh injury, while out-of-favour striker Mariano Diaz picked up an unspecified problem last week but was never likely to start.
However, Ancelotti has received a boost with the timely return of Ballon d'Or winner Karim Benzema, who missed out against Osasuna due to muscular fatigue but will travel to Anfield with the rest of the squad.
In Benzema's absence, Federico Valverde and Marco Asensio scored second-half goals in Saturday's 2-0 win over Osasuna, after which Ancelotti declared that both Kroos and Benzema would be fit for the meeting with Liverpool.
"We're fine. We had a last-minute problem with Tchouameni, who had a stomach problem. Toni and Karim are going to be fine for Tuesday's game and we arrive with good dynamics," Ancelotti told reporters.
"We have some problems but little by little the players will recover. I think we'll go into Tuesday's game with all the confidence in the world."
Kroos has failed to recover in time, though, and his absence could open the door for former Arsenal loanee Dani Ceballos to start, while Eduardo Camavinga will deputise for Tchouameni.
Ancelotti also has the option of deploying Valverde in a central position, but the versatile Uruguayan continues to excel on the right-hand side and scored his 12th goal of the season against Osasuna.
As a result, Valverde will expect to support Benzema and Vinicius Junior - who scored the winning goal in last year's Champions League final against Liverpool - for the first leg at Anfield.
Ancelotti confirmed over the weekend that Dani Carvajal would return at right-back for Nacho Fernandez, while David Alaba will likely start on the left as Eder Militao and Antonio Rudiger pair up at the back.
First-choice goalkeeper Thibaut Courtois, who made nine saves in last year's final, recovered from a hamstring in time to start against Osasuna and is in the squad, as are youngsters Alvaro Rodriguez, Mario Martin and Sergio Arribas.
Following Tuesday's first leg, Real Madrid will welcome Liverpool to the Bernabeu for the second leg on March 15.
Real Madrid squad in full:
Courtois, Lunin, Lopez; Carvajal, Vazquez, Odriozola, Vallejo, Nacho, Militao, Rudiger, Alaba; Modric, Camavinga, Valverde, Ceballos, Martin, Arribas; Rodrygo, Asensio, Benzema, Hazard, Vinicius Jr, Rodriguez