Abstract Background: Three-dimensional (3D) cell culture models are more extensive to be better models than two-dimensional (2D) cell culture models due to enriched cellular signaling pathways, cell to-cell contact, more representative of tissue morphology, and predictive of drug responses in vivo. Different drugs screening and various biological responses between 2D and 3D cell culture models have been reported. However, very little information is available on cell function and/or drug susceptibility caused by a difference in methodology of three dimensional culture. Here, we compared drug responses of various cancer cells against distinctive anti-cancer drugs when grown in monolayer, scaffold 3D culture (Matrigel, NanoCuluture Plate (NCP)) or scaffold-free 3D culture models (ultra-low attachment round bottom plate), and also confirmed which method is suitable for high-throughput screening for robust three-dimensional screening model. Method: We examined drug sensitivities of different lung cancer (A549, H226, H1650), breast cancer (SKBR-3, JIMT1), and prostate cancer (DU145) cell lines under monolayer or 3D cell culture conditions against several anti-cancer agents i.e. molecular targeted agent (Gefitinib, Afatinib, Temsirolimus, Vemurafenib, Vismodegib, Sorafenib), demethylation agent (Azacytidine, Decitabine), anti-microtubule agent (Docetaxel), alkylating agent (Cisplatin, Irinotecan), antimetabolite agent (5-FU, Gemcitabine, Methotrexate). The conditions of these assays are optimized for each culture method. Cell proliferation was measured by ATP assay. Furthermore, we analyzed the biological/ morphological differences among 3D culture methods (scaffold or scaffold-free culture models). Result: Large variations in drug responses were observed among the different cell culture models. Cells grown in 3D scaffold culture model (NCP) were more drug sensitive than other cell culture models. Especially docetaxel, 5-FU, and methotrexate showed clear growth inhibition in three dimensional scaffold culture models (NCP) than scaffold-free culture models (round-bottom). Conclusion: In this study, we showed that scaffold 3D culture is appropriate for anti-cancer drug-susceptibility test. We also proposed that NanoCuluture plate which is 3D scaffold culture model, can be used as a sophisticated high-throughput three-dimensional drug screening model. Citation Format: Norio Masuda, Atsushi Mizuno, M.Mamunur Rahman, Kazuya Arai, Manabu Itoh. Effect of scaffold on drug sensitivity of multicellular spheroids: Which method is close to in vivo and suitable for HTS. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 303. doi:10.1158/1538-7445.AM2015-303