Abstract: OBJECTIVE To establish a determination method of 3 macrolide antibiotics (erythromycin A, dirithromycin, spiramycin Ⅰ), and it's metabolite (anhydroerythromycin A, erythromycin A ene alcohol ether, erythromycylamine, neospiramycin Ⅰ) in plasma using QuECHERS followed by HPLC-MS. METHODS The macrolide antibiotics in samples were extracted with acetonitrile. The plasma was directly purified by QuECHERS pretreatment method. MgSO₄ and sodium acetate anhydrous were added into plasma sample for precipitating protein and salting out stratification. Then, MgSO₄ and PestiCarb were used for dehydration and removing impurity. The upper layer was evaporated to dryness under a stream of nitrogen and the residue obtained was redissolved in methanol. Three macrolide antibiotics were separated by Eclipse Plus C₁₈ chromatographic column. Electro spray ionization was used to analyze these macrolide antibiotics by using positive ion mode. Multiple reaction monitoring was used to monitor ions. Internal standard method was used to quantitative calculation. RESULTS Three macrolide antibiotics showed good linear relationship in the range of 4-900 ng·mL^-1(r>0.999). The quantitation limits was <10 ng·mL^-1. At three levels, the average extract recoveries were 81.45%-96.82%, and the intra-day and inter-day precisions were respectively lower than 5.00% and 10.00%, respectively. CONCLUSION The analytical method of 3 macrolide antibiotics in plasma using QuEChERS and HPLC-MS is simple, rapid, sensitive and specific. The analytical method is suitable to quantitate macrolide antibiotics and their metabolites in plasma.