<p>Melatonin, an indoleamine, is a chemical present in trace levels in different organisms. Its significance in the regulation of biological functions and therapeutic monitoring necessitates highly specific and sensitive detection methods. Despite the widespread usage of immunoassay (enzyme-linked immunosorbent assay, chemiluminescent immunoassay, and radioimmunoassay) methods, they often encounter problems with specificity, cross-reactivity, and operationality. Recent advances in biosensing technologies have overcome these limitations by providing highly sensitive, real-time, and highly specific detection with low limit of detection values. Reports on metal-organic framework combined with molecularly imprinted polymers electrochemical biosensor with limit of detection of 0.18 ng/mL, gold nanoparticles/polydopamine impedimetric biosensor with the lowest reported limit of detection of 0.32 pM and a linear range between 1 and 18 pM, and graphene voltametric biosensor with limit of detection of 0.8 µM, limit of quantification of 2.9 µM, with recoveries of 98.66–102.5% outperform the commercial kits with limit of quantification ranging from 1 to 10 µM, such as radioimmunoassay kit with limit of quantification ~ 4 pM, hence their potential application in point-of-care use. This review provides a detailed comparison of immunoassays and chromatographic techniques, including method validation data and advances that improve the sensitivity and numerical performance of biosensors. Therefore, by performance metrics and comparative assessment of detection strategies, this review provides a comprehensive framework to guide the rational selection and development of analytical tools for melatonin detection.</p> Graphical Abstract <p>Different methodologies for the detection and quantification of melatonin in pharmaceuticals.</p> <p></p>