Abstract Bioanalysis describes quantitative estimation of chemicals or drug substances as well as their metabolic products in large variety of bio-samples. It is an integrated technique that has been employed in preclinical stages of drug-discovery process to further support the clinical phases of drug discovery. However, a bioanalytical method must be optimized, characterized, and validated following documented procedures according to United States Pharmacopeia (USP)/International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines to comply with the regulatory guidelines and acceptance criteria. Bioanalytical studies should therefore provide accurate and reproducible estimation of drugs or metabolites in biological samples at great level of sensitivity, selectivity, and specificity. In this chapter, various advanced techniques commonly employed in bioanalysis have been summarized. Different techniques of extraction and an array of advanced hyphenated techniques are employed for the evaluation of bioanalytes in biofluids, with improved analytical specificity and sensitivity. The applications of bioanalysis in biomedical, pharmaceutical, and other allied areas have been systematically reviewed in this chapter.