A new spectrophotometric method has been developed to accurately measure creatine levels in plasma, urine, and serum. This improved method is suitable for routine use in clinical laboratories and the pharmaceutical industry. The proposed method utilizes Alpha naphthol Sulpha Acetamide (ASA), a new azo dye, to react with creatine in order to produce an orange-colored complex that can be detected at a wavelength of 525 nm. The optimal conditions for this method have been thoroughly studied, including various factors such as pH, buffer, dye volume, sequence of addition, time, temperature, organic solvent, surfactant and interfering materials. It has been determined that the most optimal conditions for determining creatinine at room temperature are a temperature of 20°C after 5 minutes in the presence of citrate buffer pH(5) and Triton X-100 as a surfactant. The concentration range of (0.149- 3.729) µg mL-1 with ASA dye obeys Beer's law, with a molar absorptivity of 11980 L-1.mol-1.cm-1. This improved method is expected to contribute greatly to the clinical and pharmaceutical industries due to its accuracy and convenience.