•Sample preparation techniques coupled with high-performance liquid chromatography and a suitable detector.•Combining sample preparation techniques to achieve satisfactory extraction efficiency, cleanup as well as excellent preconcentration capacity.•Validating an analytical method to ensure the quality and reliability of any data produced is a fundamental step in any measurement's life-cycle.•Sample preparation techniques future. This review article compares and contrasts sample preparation techniques coupled with high-performance liquid chromatography (HPLC) and describes applications developed in biomedical, forensics, and environmental/industrial hygiene in the last two decades. The proper sample preparation technique can offer valued data for a targeted application when coupled to HPLC and a suitable detector. Improvements in sample preparation techniques in the last two decades have resulted in efficient extraction, cleanup, and preconcentration in a single step, thus providing a pathway to tackle complex matrix applications. Applications such as biological therapeutics, proteomics, lipidomics, metabolomics, environmental/industrial hygiene, forensics, glycan cleanup, etc., have been significantly enhanced due to improved sample preparation techniques. This review looks at the early sample preparation techniques. Further, it describes eight sample preparation technique coupled to HPLC that has gained prominence in the last two decades. They are (1) solid-phase extraction (SPE), (2) liquid-liquid extraction (LLE), (3) gel permeation chromatography (GPC), (4) Quick Easy Cheap Effective Rugged, Safe (QuEChERS), (5) solid-phase microextraction (SPME), (6) ultrasonic-assisted solvent extraction (UASE), and (7) microwave-assisted solvent extraction (MWASE). SPE, LLE, GPC, QuEChERS, and SPME can be used offline and online with HPLC. UASE and MWASE can be used offline with HPLC but have also been combined with the online automated techniques of SPE, LLE, GPC, or QuEChERS for targeted analysis. Three application areas of biomedical, forensics, and environmental/industrial hygiene are reviewed for the eight sample preparation techniques. Three hundred and twenty references on the eight sample preparation techniques published over the last two decades (2001–2021) are provided. Other older references were included to illustrate the historical development of sample preparation techniques.