In previous study, tumor cell under three-dimensional (3D) spheroids culture condition makes different phenotype compared to two-dimensional (2D) culture condition. To make a reliable cell line experiment, spheroid culture condition have been thought to be reasonable but there have been no clear molecular characterization about this issue. In this study, comparative proteome analysis between spheroid cell cultured using home-developed membrane ( poly(2,4,6,8-tetravinyl-2,4,6,8- tetramethyl cyclotetrasiloxane) (pV4D4), and commercial hydrophilic membrane ultra-low-attachment (ULA). Conventional 2D monolayer-cultured control cell was also analysed using high resolution LC-MS platform coupled by multiplexing peptide labeling technology. And quantitative proteome of conditioned media from each culture condition were analyzed to investigate the difference at secretory part. As a result, EGFR pathway and mitochondrial dysfunction pathway is involved in pV4D4-based spheroid culture condition compare to other culture condition. From this study, we could conclude that pV4D4-based spheroid cultured cell shows stem-like characteristics and that this 3D spheroid culture can be a useful tool to investigate cancer stem cell and its resistance to chemotherapy.