This study presents an improved spectrophotometric method for measuring catalase (CAT) activity in biological samples, characterized by simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with hydrogen peroxide (H₂O₂) and then adding a solution of ferrous ammonium sulfate (FAS) and sulfasalicylic acid (SSA) to terminate the reaction. The formation of a maroon-colored ferrisulfosalicylate complex, which has a maximum absorbance at 490 nm, indicates the presence of residual H₂O₂. This method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. The method was validated through a comparative analysis with the standard ferrithiocyanate method, showing a correlation coefficient of 0.99, demonstrating its effectiveness and reliability. The SSA-CAT assay is unaffected by different types of biomolecules and can be used in research and clinical analysis with considerable accuracy and precision. The study also evaluated the method's linearity, sensitivity, reproducibility, selectivity, and accuracy, confirming its robustness and applicability in various biological samples, including liver tissue homogenates, bacterial lysates, and urine samples. The results highlight the potential of this method for assessing CAT activity in various biological contexts.This study presents an improved spectrophotometric method for measuring catalase (CAT) activity in biological samples, characterized by simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with hydrogen peroxide (H₂O₂) and then adding a solution of ferrous ammonium sulfate (FAS) and sulfasalicylic acid (SSA) to terminate the reaction. The formation of a maroon-colored ferrisulfosalicylate complex, which has a maximum absorbance at 490 nm, indicates the presence of residual H₂O₂. This method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. The method was validated through a comparative analysis with the standard ferrithiocyanate method, showing a correlation coefficient of 0.99, demonstrating its effectiveness and reliability. The SSA-CAT assay is unaffected by different types of biomolecules and can be used in research and clinical analysis with considerable accuracy and precision. The study also evaluated the method's linearity, sensitivity, reproducibility, selectivity, and accuracy, confirming its robustness and applicability in various biological samples, including liver tissue homogenates, bacterial lysates, and urine samples. The results highlight the potential of this method for assessing CAT activity in various biological contexts.