Alli, who suffered a hamstring injury earlier this month, missed the 2-0 victory at Huddersfield, and Pochettino said there was no chance of the England international facing the Spanish side.
The midfielder suffered the injury during England's Nations League defeat to Spain earlier this month and missed the matches with Liverpool and Inter Milan.
Although he was back again as a substitute against Brighton last weekend and scored in the League Cup against Watford on Wednesday, he has suffered an aggravation of the injury.
Pochettino said it was too early to evaluate the injuries to Jan Vertonghen and Mousa Dembele, who were both substituted at half-time in the win at the John Smith's Stadium.
"It is not clear if they are muscle (injuries) or knocks," said the Spurs manager, who used the injuries to emphasise his unhappiness at criticism of his squad rotation.
"The season is a massive challenge for everyone. A lot of teams have the same problem. The players are not machines. We want to push and push and push them -- it is not easy.
"No-one helps, all want that in every single game play the same players," he added, highlighting the criticism he received for resting players.
But the Spurs manager said muscle injuries were a risk facing a number of teams.
"I think the teams that had players involved in the World Cup are going to have this kind of problems," he added.
(Reporting by Simon Evans; editing by Clare Fallon, Neville Dalton)
