Pharmaceutical investigators are searching for preclinical models closely resembling the original cancer and predicting clinical outcome. This study compares drug response of three in vitro 3D-drug screening models with different complexity. Tumor cell line spheroids were generated from the cell lines Caco-2, DLD-1, COLO 205, HT-29 and HCT-116, and treated with clinically relevant combination therapies, namely 5-FU/oxaliplatin (FO), 5-FU/irinotecan (FI) and the molecular drugs Cetuximab, Trastuzumab, Vorinostat and Everolimus. Treatment results were compared with spheroids originated from tumor cell lines (Caco-2, DLD-1) co-cultured with stromal cells (PBMCs, cancer-associated fibroblasts of colorectal origin) and spheroids directly prepared from colon cancer tissues. Different microenvironment compositions altered the tumor cell line spheroid response patterns. Adding PBMCs increased resistance to FO treatment by 10-15% in Caco-2 and DLD-1 spheroids but decreased resistance to FI by 16% in DLD-1 spheroids. Fibroblast co-cultures decreased resistance to FI in Caco-2 spheroids by 38% but had no impact on FO. Treatment of colon cancer tissue spheroids revealed three distinct response pattern subgroups not detectable in 3D cell lines models. The cancer tissue spheroid model mimics both tumor characteristics and the stromal microenvironment and therefore is an invaluable screening model for pharmaceutical drug development.