Extraction of collagen from muscles of cultured catfish (Clarias gariepinus × C. macrocephalus) with the aid of pepsin digestion was investigated using a statistical tool. Fractional factorial design (FFD) was applied to evaluate the effects of eight process parameters: acetic acid concentration, acid extraction time, acid extraction temperature, acetic acid to muscles ratio, NaOH concentration, NaOH to muscles ratio, NaOH treatment time, and stirring speed. Contribution of every parameter in influencing the extraction efficiency was evaluated and factors that significantly affected the extraction were elucidated by employing experimental design and analysis of variance in FFD. The result of first order factorial design showed that acetic acid concentration, acid extraction time, acid to muscles ratio, and stirring speed had significant effect (P < 0.05) to the yield of pepsin soluble collagen (PSC) obtained at the end of the experiment. Effects of these process factors on the efficiency of collagen extraction were investigated, and are discussed in detail. Optimum conditions were found at 0.5 M acetic acid, 16 hr extraction period, solvent to muscles ratio at 25 ml/g, and stirring speed of 400 rpm, resulting in yield of PSC as high as 211.49 ± 15.51 mg/g.