Abstract A chemically-defined protein-free medium (FMX-Turbodoma) has been improved for the production of monoclonal IgA antibodies by hybridoma cells, using a systematic method. Cell growth rate, IgA production, activity and molecular weight pattern have been used as optimization criteria. Of potentially important supplements, glucose, glutamine, Pluronic acid F-68 as well as several amino acids had significant beneficial effects. A determination of amino acids profiles via HPLC analysis allowed the formulation of a balanced medium. Unbalanced supplementations of amino acids were found undesirable because of the toxicity of some amino acids at high concentration. Compared with the basal medium, the maximum viable cell and final IgA concentrations in the final version of the protein-free medium were increased by 130% and 700%, respectively, whereas the IgA molecular weight pattern and in vitro activity were not affected. The IgA production was even higher than in a serum-containing medium (RPMI 1640 + 10% FCS) and the price of the protein-free medium is about 20% of this serum-containing medium. This makes such a protein-free medium very convenient for laboratory and large-scale production.