De la Fuente selected an experimental XI at the London Stadium, with Dani Vivian making his debut as a starter, while Alex Remiro and Pau Cubarsi were also handed their first appearances for the national side off the bench.
Crystal Palace defender Daniel Munoz scored the only goal of the contest, with the 27-year-old registering in the 61st minute in the English capital.
The defeat ended an eight-game winning run for Spain, who will be back in action against Brazil on Tuesday night.
However, De la Fuente took the positives from the contest, with the manager looking to the future.
"You learn more from defeats than from victories," Marca quotes De la Fuente as saying. "We wanted to test some things, see how certain players performed, but we keep working and improving.
"The debutants all have great futures, and we need them to have the opportunity in these games, so that when we play for points, they already have experience."
De la Fuente is expected to name a stronger XI for the clash with Brazil on Tuesday, as the likes of Unai Simon, Dani Carvajal, Robin Le Normand, Rodri and Dani Olmo did not feature against Colombia.
Spain, as it stands, have one friendly in the diary for June, facing Andorra, before beginning their Euro 2024 campaign against Croatia in the middle of the month.
Barcelona's 17-year-old centre-back Cubarsi came off the bench in the 83rd minute of the clash, and the youngster revealed his delight at making his debut.
"I'm very proud of my debut as it was a dream I've had since I was a kid. I'm grateful for the coach's trust and I will try to make the most of it by giving my best. I always try to be me and play calmly," he told reporters.
Cubarsi will be bidding to make the squad for this summer's European Championship, and strong performances for Barcelona for the remainder of the campaign could see him on the plane to Germany.
Gavi and Pedri are notable absentees from the current Spain squad, with the former set to miss Euro 2024 due to the serious knee injury which he picked up towards the end of last year.