Chromatography columns with longer length and more theoretical plates per unit have more resolving power, but it is time consuming and high usage of costly solvents. The particles in the stationary phase play a very important role in resolution of the peaks. Less the particle size more is resolving power of stationary phase. However, backpressure becomes very high, and, in this context, strong pumps have been introduced, and these developments have resulted in faster and better resolution and explain why high-performance liquid chromatography (HPLC) has emerged as the most popular, powerful, and versatile form of chromatography. The major components in HPLC includes solvents, sampler, pump, column and detectors have been aptly described in this chapter. The application of HPLC includes assay of pharmaceutical products monitoring drugs and metabolites in body fluids, detection of drugs of abuse, determination of additives in food and beverages, quality control of polymers, plastics, and resins. At the outset of chromatography, HPLC was commonly used for pesticide residues analysis, but with introduction of mass spectrometry HPLC is now on the backfoot.