The accurate determination and quantification of praziquantel are essential for optimizing its therapeutic effectiveness in treating schistosomiasis and neurocysticercosis, two significantly neglected tropical diseases. Its challenging physicochemical profile, extensive metabolism, and stereochemical complexity requires robust analytical methods for reliable quantification in clinical, veterinary, and pharmaceutical samples. This review provides a comprehensive and critical evaluation of analytical strategies used for PZQ determination, spanning fluorometric and radiometric assays, HPLC–UV, LC–MS, LC–MS/MS, and enantioselective chromatographic approaches. Particular emphasis is placed on the evolution toward highly sensitive LC–MS/MS methods and their alignment with contemporary regulatory expectations, including ICH M10 requirements. These advancements have significantly improved sensitivity, specificity, and reproducibility, which are crucial for pharmacokinetic, pharmacodynamic, and bioequivalence studies. Enantioselective methods for distinguishing PZQ enantiomers and metabolites are discussed. The aim of these innovations is to increase praziquantel bioavailability, improve patient adherence, and support its continued use in mass drug administration programs. Finally, the review highlights implementation challenges in resource-limited settings and proposes analytical models to expand global bioanalytical capacity. Together, these insights provide a structured foundation for selecting and developing high-quality, regulatory-compliant analytical methods for PZQ.