The most common analytical method for detecting, measuring, and separating components in complicated mixtures is high-performance liquid chromatography. This review provides a comprehensive demonstration of HPLC, including its basic principles, instrumentation, operational modes, and applications in the field like pharmaceuticals, environmental analysis, food quality control, and biotechnology. Emphasis is placed on the different HPLC modes such as size-exclusion chromatography, reversed-phase, ion- exchange C alongside method development, normal-phase optimization validation. The article also discusses recent advancements and the integration of HPLC with modern detection techniques such as mass spectrometry (HPLC-MS) for enhanced analytical capabilities.