Abstract: An effective method was established and verified for the detection of clarithromycin and its relative substances J, M, N and K by HPLC. Clarithromycin relative substances sample was separated by isocratic elution with a Purospher STAR LP RP-18e C18 column and with the mobile phase 0.067 mol/L KH<sub<2</sub<PO<sub<4</sub< solution (pH 4.0)/acetonitrile=60/40, the flow rate of 1.2 mL/min, the column temperature 35℃ and detection wavelength 205 nm, and the separation degrees were greater than 1.2. Analysis results show that, the method can provide good linearity (<i<R</i<<0.999) for all relative substances at the concentration range of 20~240 μg/mL, the average recovery rates keep at 87.80%~121.17%, its operation is simple and efficient, and can be able to simultaneously detect J, M, N and K in clarithromycin.