Statistically designed experiments were used in developing a low serum medium (LSD) for the production of a diagnostic monoclonal antibody (MAb) against congenital adrenal hyperplasia. Hybridoma 192 was used for producing the antibody. A two-level half-fractional factorial design was used for screening six components (Minimum Essential Medium Eagle (MEM) amino acids, 2-mercaptoethanol, ethanolamine, ferric citrate, zinc sulfate and sodium selenite). The experimental design was then augmented to central composite design. The basal Dulbecco’s modified Eagle’s medium (DMEM) (containing 4 mM L-glutamine, 1% antibiotic-antimycotic agent) supplemented with 0.4% by volume fetal bovine serum, 311.8 mM ferric citrate, 17.3 nM sodium selenite and 4.5 mM zinc sulfate, was found to support the growth of the hybridoma. Specific cell growth rate in the low serum medium (LSD) was slightly lower than in the control medium (i.e. the above specified basal DMEM supplemented with 2% fetal bovine serum). The specific growth rates in LSD and the control media were 0.037 ± 0.004/h and 0.048 ± 0.003/h, respectively. Nevertheless, the specific MAb production rate for cells in LSD was comparable to that in the control culture (0.004 ± 0.001 pg/cell.h versus 0.005 ± 0.000 pg/cell.h). The bioactivity assay for the antibody produced in the LSD gave results that were comparable to the control culture, revealing no structural changes owing to the new medium. The new medium formulation effectively reduced the medium cost by up to 87% and enhanced the cell specific MAb production rate.