Abstract: HPLC was applied to the determination of bacitracin A in fermentation liquor. The crude extract obtained by centrifuging fermentation liquor of bacillus licheniformis, was extracted with trichloroacetic acid. After centrifugation, the supernatant was separated by gradient elution on Bechman C18 column with mixtures of acetonitrile and potassium dihydrogen phosphate buffer solution in various ratios as mobile phase. Wavelength of 220 nm was chosen for UV-detection for bacitracin A. Linear relationship between values of peak area and mass concentration of bacitracin A was obtained in the range of 1.0-15.0 g·L-1, with detection limit (3S/N) of 0.08 g·L-1.Test for recovery was made by addition of bacitracin A standard at two concentration levels of 7.37 and 11.01 g·L-1, values of average recovery obtained were 98.1% and 97.4%, respectively.