The NS0 myeloma cell line is often used for the production of monoclonal antibodies (Mabs) and other recombinant proteins. The growth of these mammalian cells in a protein-free media has several advantages including cost, safety, consistency, efficiency and regulatory approval. However, the adaptation of the NS0 myeloma cell line so as to grow in a protein-free medium is poorly understood. In order to better understand this process, we applied proteomic techniques, specifically two-dimensional electrophoresis (2DE) and mass spectrometry (MS), to identify the key pathways involved in this adaptation. The analysis of changes in protein expression between the host myeloma cell line and two recombinant NS0 cell lines expressing different humanized Mabs has primarily revealed changes in proteins associated with carbohydrate metabolism, energy production, protein synthesis and folding, membrane transport and cell proliferation. Other factors that may be involved in the adaptation of NS0 myeloma cells to a protein-free medium are reported.