Background <p>Melatonin is an important endogenous hormone widely studied for its therapeutic potential, requiring reliable and sustainable analytical methods for its quantification in biological matrices. Conventional analytical techniques often involve complex procedures and environmentally hazardous solvents, highlighting the need for a simple and green bioanalytical approach.</p> Objective <p>The present study aimed to develop and validate a simple, sensitive, and environmentally benign bio-analytical method for the quantitative determination of melatonin in human plasma.</p> Methods <p>Melatonin was extracted from human plasma using a liquid-liquid extraction technique and analyzed at 225 nm. The method was validated for linearity, precision, accuracy, recovery, selectivity, ruggedness, and robustness in accordance with bioanalytical validation principles. Environmental sustainability was assessed using Analytical GREEnness (AGREE) and Blue Analytical Greenness Index (BAGI) tools.</p> Results <p>The method demonstrated good linearity over a concentration range of 2-10 µg/mL with a correlation coefficient (r²) of 0.997. Intra- and inter-day precision values were within acceptable limits, indicating good repeatability and reproducibility. Accuracy studies showed satisfactory recovery across all quality control levels. The extraction procedure was efficient and consistent, with no endogenous interference observed at the detection wavelength. Ruggedness and robustness studies confirmed the stability of the method under minor variations. AGREE and BAGI assessments indicated a high greenness score.</p> Conclusion <p>The developed UV spectrophotometric method is simple, reliable, and environmentally sustainable for the quantification of melatonin in human plasma and can be effectively applied for routine bioanalytical analysis.</p> Graphical Abstract <p></p>