This article compares the benefits and drawbacks of each approach while reviewing recent advancements in bioanalysis sample preparation methods. It also provides an update on fundamental concepts, theories, applications, and automation opportunities. Because every biological matrix has its own special difficulties and complexity, sample preparation is thought to be the bottleneck stage in bioanalysis. In the last ten years, new sample preparation methods for bioanalysis have developed quickly. In the preclinical and clinical phases of drug development, it is crucial to create reliable bioanalytical method(s). Consequently, it is widely acknowledged that sample preparation A number of excellent review papers have recently been published in the literature that address various scientific and technical facets of bioanalysis. The use of bioanalysis in the pharmacokinetic/pharmacodynamic characterization of novel chemical entities, beginning with their finding and continuing through their market authorization, is now generally acknowledged. Physico-chemical and biological technologies used in bioanalysis, including mass spectrometry, immunoassay, and chromatography. Liquid chromatography-mass spectrometry is a method used in labs for the qualitative and quantitative study of drug substances and metabolites. It makes use of liquid chromatography/RP HPLC. The current review concentrated on different extraction methods, including protein precipitation, solid phase extraction, and liquid-liquid extraction, all of which are crucial for sample preparation and RP HPLC sample detection.