This study presents a novel spectrophotometric method for determining catalase (CAT) activity in biological samples, characterized by simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of hydrogen peroxide (H₂O₂) for a specified period. A solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is then added to terminate the enzyme activity, forming a maroon-colored ferrisulfosalicylate complex. The complex has a maximum absorbance of 490 nm, allowing for the quantification of CAT activity. This method eliminates the need for concentrated acids, making it safer and easier to handle. A comparative analysis with the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's effectiveness and reliability. The method is suitable for use in research and clinical analysis with high accuracy and precision. The study also evaluated the method's linearity, sensitivity, reproducibility, and accuracy, finding it to be reliable for measuring CAT activity. The method was applied to assess CAT activity in liver tissue homogenates, bacterial lysates, and urine samples, showing its versatility. The results indicated that CAT activity is significantly lower in smokers compared to non-smokers, suggesting that tobacco smoking reduces CAT activity in erythrocytes. The method is environmentally friendly and aligns with green chemistry principles. The study concludes that the proposed method is a fast, precise, and versatile tool for measuring CAT activity in various biological samples.This study presents a novel spectrophotometric method for determining catalase (CAT) activity in biological samples, characterized by simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of hydrogen peroxide (H₂O₂) for a specified period. A solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is then added to terminate the enzyme activity, forming a maroon-colored ferrisulfosalicylate complex. The complex has a maximum absorbance of 490 nm, allowing for the quantification of CAT activity. This method eliminates the need for concentrated acids, making it safer and easier to handle. A comparative analysis with the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's effectiveness and reliability. The method is suitable for use in research and clinical analysis with high accuracy and precision. The study also evaluated the method's linearity, sensitivity, reproducibility, and accuracy, finding it to be reliable for measuring CAT activity. The method was applied to assess CAT activity in liver tissue homogenates, bacterial lysates, and urine samples, showing its versatility. The results indicated that CAT activity is significantly lower in smokers compared to non-smokers, suggesting that tobacco smoking reduces CAT activity in erythrocytes. The method is environmentally friendly and aligns with green chemistry principles. The study concludes that the proposed method is a fast, precise, and versatile tool for measuring CAT activity in various biological samples.