Emerging evidence suggests that the nutrient availability in the tumour cell microenvironment and the dimensionality of in vitro tumour cells impact cell proteome and drug responsiveness. An improved physiological medium, called Melbourne Medium (MM), has been developed most recently by our research group. MM was designed to emulate low molecular weight components of human plasma, so it is a more physiologically relevant medium compared to conventional medium such as DMEM (CM). Global proteomics experiments were conducted to compare the protein profiles of MCF-7 and MDA-MB-231 breast cancer cells cultured in MM versus CM and in different experimental conditions (3D versus 2D).