Objectives: The purpose of this paper was to validate an alternative method for the determination of papain activity in pharmaceutical formulations using a low molecular weight substrate called N-Benzoyl-DL-arginine-ρ-nitroanilide. Methods: The assessment of enzymatic activity is based on the amount of ρ-nitroaniline released over time formed during the hydrolysis of this substrate caused the active papain, estimated colorimetrically at 405 nm at pH 6.0 and 40 o C. The method was validated according to the United States Pharmacopeia guidelines. Parameters such as accuracy, precision, linearity, limit of detection and limit of quantification were estimated. Prototype-solid, semi-solid and liquid-formulations were prepared and assayed using this technique. Results: The results revealed linearity established as R= 0.9978, precision values of 1.68% and accuracy of 94.15%. The method also holds a limit of detection and limit of quantification of 123.98 USP.mL-1 and 413.27 USP.mL-1 respectively. No interference was observed when the prototype formulations were assayed. Conclusion: The results indicated that such methodology is an alternative, reliable and adequate method to determine the papain activity in pharmaceutical formulations.