Objective: The primary purposes of this research were to develop and validate a novel, accurate, sensitive, and repeatable bioanalytical method for determining amikacin in human plasma employing UPLC-MS/MS. Methods: The bioanalytical procedure of amikacin involved a BEH C18 UPLC column as a stationary phase, with an employed mobile phase consisting of 0.1% v/v formic acid and acetonitrile (85:15 v/v). The flow rate was set at 0.1 ml/min, and the column temperature was kept at 30 °C. Kanamycin was selected as an internal standard. Amikacin and kanamycin were determined at mass-to-charge ratios (m/z) of 585.9>162.9 and 484.67>162.83, respectively. The amikacin bioanalysis method in the plasma matrix at the optimum separation condition was validated by determination of selectivity, linearity, accuracy, precision, recovery, carry-over, matrix effect, and stability. Results: The optimum conditions of the sample preparation procedure were obtained through liquid-liquid extraction using trichloroacetic acid, followed by vortex mixing for one minute and centrifugation at 10,000 rpm for five minutes. Ten µl of supernatant was collected and injected into the system. A linear response was achieved in the 1.0-150.0 µg/ml range with R2 0.9997. Accuracy and precision met the requirements with % differences and coefficient variation at all concentration levels less than 15% and at the LLOQ level (1 μg/ml) less than 20%. The validated analytical method of amikacin in plasma is required for therapeutic monitoring in patients. The data would be valuable for determining or adjusting amikacin doses to enhance patient safety. Conclusion: A bioanalytical method was developed and validated for determining amikacin in human plasma by UPLC-MS/MS. The method selectivity, linearity, accuracy, precision, recovery, carry-over, matrix effect, and stability were performed. © 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd.