Four hybridoma cell lines (C4B, 10C2G5, 6C5F4C7, 2D10G11) were adapted to grow in serum-free conditions. Cell proliferation, viability, and antibody production in Nutridoma SP and Ex-Cell HSF 610 media were evaluated and results compared with those obtained using DMEM containing 10% fetal bovine serum (control medium). Clone C4B showed the best IgG productivity in control medium, but viability and number of cells per milliliter were similar for the three media. For clone 10C2G5, the highest values of cell viability were obtained with both control medium and Nutridoma SP; no significant differences in IgG yields and number of cells/mL were observed among the three media. Clone 6C5F4C7 provided no significant differences when grown in the two serum-free media and in control medium. Clone 2D10G11 showed the best IgG productivity in control medium and in Ex-Cell HSF 610, even if Ex-Cell HSF 610 provided the lowest number of cells/mL; no significant differences among the three media were obtained for viability. Purified antibodies produced from each hybridoma cell line grown in serum-free media showed a higher degree of purity than those produced by the same cell lines grown in control medium. Purified MAbs were also titrated by ELISA to test MAb-antigen affinity.