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This paper describes the development and validation of a most economical and sensitive isocratic stability indicating HPLC method for the assay of recombinant human insulin (RHI) from yeast origin (Hansenula polymorpha). The method employs a Thermo Biobasic, C-18 (250 x 4.6 mm, 300 A°, 5 µm) column with a mobile phase of acetonitrile - potassium dihydrogen phosphate ( pH 2.5; 50 mM) (35:65, v/v) and UV detection at 242 nm. Factorial design was used to facilitate method development. Buffer pH and concentration of acetonitrile were considered as the independent variable to study capacity factor of RHI. Sixteen experiments were undertaken, and a quadratic model was derived for the capacity factor of RHI. The method produced well defined, sharp peaks having lower tailing factor (1.114). A linear response with a correlation coefficient (r2) of 0.999 was observed over the range of 0.025 - 2.0 IU mL-1 …