In this study we analyzed the proteomic profiles of H460, and MCF7 3D tumor spheroids grown either in standard medium (3D_SM), or in FBSlow conditions (3D_FBSlow) and compared them to their relative 2D cultures (2D). Standard medium consisted of DMEM/F-12 supplemented with 0.5% Glucose, 2.5mM L-Glutamine, 2% B-27, 5μg/ml Heparin, 20μg/ml Insulin, 20ng/ml EGF, 20ng/ml Recombinant Human bFGF, 0.1% Bovine Serum Albumin (BSA) and 1% (v/v) of penicillin/streptomycin 100U/ml. FBSlow medium consisted of customized nutrient-restricted RPMI or DMEM culture media supplemented with only 2% FBS.