The main impact of bionalysis in the pharmaceutical industry is to obtain a quantitative measure of the drug and its metabolites. The purpose is to perform the pharmacokinetics, toxicokinetics, bioequivalence and exposure response like pharmacokinetic/pharmacodynamic studies. Various bioanalytical techniques are performed in bioanalytical studies such as hyphenated techniques, chromatographic techniques, and ligand binding assays. This review extensively highlights the role of bioanalytical techniques and hyphenated instruments in assessing the bioanalysis of the drugs. Many scientific endeavors are dependent upon accurate quantification of drugs and endogenous substances in biological samples; the focus of bioanalysis in the pharmaceutical industry is to provide a quantitative measure of the active drug and/or its metabolite(s) for the purpose of pharmacokinetics, toxicokinetics, bioequivalence and exposure–response (pharmacokinetics/pharmacodynamics studies). Bioanalysis also applies to drugs used for illicit purposes, forensic investigations, anti-doping testing in sports, and environmental concerns. Separation process involved between biomolecules like proteins, amino acids, sugars, cells, tissues, Microorganisms which can separated by using various techniques like electrophoresis, Cytometry, Elucidation are dealt under this topic.