Elche native Saul Niguez opened the night's scoring in the 24th minute with a leaping header off Dani Carvajal's long diagonal cross that bounced past Croatia goalkeeper Lovre Kalinic.
Spain coach Luis Enrique has admitted he made a mistake to not select Celta Vigo forward Iago Aspas in his initial squad after seeing him in training. In the 57th minute, Spain captain Sergio Ramos headed in his 14th global goal for the team's fifth on the night.
Croatia could do little to stop the Spanish onslaught as the World Cup finalists suffered their worst ever defeat in team history.
Rodrigo scored after slipping past the Croatian backline on a through-ball from Asensio in the 49th minute. This will be the team's first Nations League match.
The Real Madrid star has now lost all three matches he's played against Spain in his career.
Spain's next match takes place in exactly a month, when they visit Cardiff for a friendly four days before hosting England in the Nations League.
Luis Enrique's charges got their campaign underway over the weekend with a 2-1 win over England at Wembley Stadium.
Croatia drew 1-1 with Portugal on Thursday night in their first game since losing to France in the final in Russian Federation, while Spain will be on home soil under new boss Luis Enrique for the first time following his appointment.
Croatia will also be back in action in October, but face another tough test against the Three Lions on their return to the pitch.