Calibration of an instrument is the demonstration that the instrument or device produces results within specified limits by comparison with those produced by reference or traceable standard over an appropriate range of measurement. Calibration of HPLC is very important process for initiating the NABL process. The parameters for calibration include pressure test, drift and noise, column oven and sample cooler, pump by flow rate accuracy and measurement, pump by gradient flow measurement, and UV-Vis/PDA detector by reference energy check. Recommended frequency for these tests is done quarterly and half yearly. A complete description of the calibration has been provided in this chapter with the tabulated format.