Abstract: The cytostatic effects of 5-fluorouracil, oxaliplatin, and 4-(5-methyl-5H-[1,3]dioxolo[4,5-f]-indol-7-yl)-3-(5-fluoro-1-benzofuran-3-yl)-1 H-pyrrole-2,5-dione (9-ING-41), a new glycogen synthase kinase-3 inhibitor, were evaluated in 2D and 3D models of HT-29 colorectal adenocarcinoma cells. The HT-29 cells cultured in 3D were resistant to the test drugs, while in the case of the 2D model, not only cytostatic but also cytotoxic effect was observed The highest cytotoxic effect was detected upon the addition of 9-ING-41, at high concentrations of which the cell viability decreased to 50% after 3 h of incubation. The results indicate that 3D cultures reproduce the tumor structure and drug resistance more accurately.