Objective: A simple, precise, and accurate spectrophotometric method has been developed to determine Ampicillin in pharmaceutical formulations. Methods: The proposed method, based on the carboxylic acid group reaction, present in Ampicillin with a mixture of KIO3 and KI, form a yellow-colored product in an aqueous medium. The response was allowed to proceed at 25±1 °C, and absorbance measured after 5 min against a reagent blank prepared simultaneously using a UV-Vis spectrophotometer. The parameters verified were specificity, linearity, linearity range, accuracy, precision, detection limit, quantitation limit, robustness, and ruggedness. Results: The yellow-colored product was measured at 352 nm against the reagent blank using UV–Vis spectrophotometer. The linear dynamic range of concentration was 0.25–2.5 µg/ml with a correlation coefficient of 0.9999. The LOD, LOQ values to be 0.086 and 0.261 µg/ml, respectively, for the proposed method. The percentage of recoveries was 98.27–100.89% with an acceptable relative standard deviation (±2%). The robustness and ruggedness values were excellent. Conclusion: The ICH guidelines for pharmaceuticals and human use were followed and applied to validate the proposed method. The method was compared with available literature and found similar results that confirmed the reliability and effective way for Ampicillin's determination.