The 18-year-old came off after falling awkwardly in a challenge and was replaced by Andy King after 50 minutes.
Ampadu has been in fine form for the Welsh this season, especially in their 4-1 win over Ireland.
But he has found game-time hard to come by at Chelsea as Maurizio Sarri continues to overhaul their midfield with new boys Jorginho and Mateo Kovacic breathing fresh energy into the middle of the park.
Ryan Giggs has one eye on Tuesday's clash with Ireland in Dublin, where Wales can keep alive their hopes of Nations League promotion and put the Irish in danger of relegation to League C.
Ampadu was class against the Irish in Cardiff and bossed the middle of the park before being substituted.
Maurizio Sarri has overlooked Ampadu in the Premier League so far this term, after freshening the Blues' midfield up with the signings of Jorginho and Mateo Kovacic.
Not that the Rastafarian-haired teen star could do anything to stop the Spanish running Wales ragged in Cardiff.
Two goals from Dortmund forward Paco Alcacer, a contentious Sergio Ramos header and another header from Marc Bartra gave Spain a comfortable lead.
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