Chromatographic conditions for isocratic HPLC determination in reverse phase and hydrophilic modes of a number of widely used antibiotics are described. In particular, examples are given of the group separation of sulfamides, quinolones and cephalosporins, as well as a set of antibiotics used in veterinary medicine, including nitroimidazoles, lincosamides, tetracyclines, sulfonamides and quinolones. It is argued that the widespread use of gradient elution for HPLC determination of antibiotics (to the detriment of the more practical isocratic approach) is mainly due to the use of insufficient quality stationary phases, as well as stationary phases with suboptimal selectivity. The results are illustrated with real examples of HPLC separations developed by specialists of the IP company Sychev KS / I.B.S.