A highly simple and sensitive catalytic spectrophotometric method for the determination of tiopronin based on its inhibitory effect on the hemoglobin-catalyzed the reaction of H2O2 and acid chrome blue K(ACBK) was developed. The concentration of tiopronin is linear with the percentage inhibition (I%) of system under the optimal experimental conditions. The calibration graph is linear in range 0.05 to 2 mg/L with the detection limit of 8.5μg/L. The relative standard deviation was 4.1 % for 11 determinations of 1.5 mg/L. This method can be used for the determination of tiopronin in pharmaceutical preparations with satisfactory results.