The two clubs will meet in their first leg tie at Juventus Stadium on 13 February in one of the more intriguing match-ups of the competition.
However, the reigning Serie A champions will be without Matuidi after the Frenchman limped out during Juventus' emphatic 7-0 win over Sassuolo on Sunday (4 February).
Suffering from a "muscular issue", Matuidi came off after just 26 minutes. But it did not affect the rampant Bianconeri who remained on league leaders Napoli's trail with just one point separating the two teams.
Following the game, Allegri confirmed that Matuidi will not be taking part against Spurs, while giving an update on Claudio Marchisio and Paolo Dybala.
"Blaise Matuidi seems to have a muscular issue, but we have players like Claudio Marchisio and Dybala who are getting closer to fitness. Matuidi won't be there against Tottenham," Allegri told Mediaset Premium via FourFourTwo.
"Many players are beginning to reach their ideal form. It was important to win so we could definitively break away from [Serie A's] chasing pack, who are now 14 points away.
"There is no secret to our success, we simply have lads with good values who want to win what feels like their first Scudetto. We want to stay on Napoli's tails going into the sprint finish."
Dybala in particular, would be a big blow to Juventus should he miss out with the Argentine forward having not featured since a 1-0 win over Cagliari back in early January.
Meanwhile, Tottenham were fortunate to leave Anfield with a crucial point after they responded to an injury-time goal from Mohamed Salah to salvage a 2-2 draw against Liverpool on Sunday.
Despite missing an earlier penalty, Harry Kane was able to bury a second penalty after Virgil van Dijk's challenge on Erik Lamela in the 93rd minute.