A sensitive and selective analytical method for 7 macrolides determination by LC-tandem mass spectrometry (LC-MS/MS) in fish and shrimp was developed. Samples were extracted by liquid-liquid extraction steps using 70% acetonitrile. The positive ionization produced the molecular related ions, $(M+2H)^{2+}$, at m/z 422.3 for spiramycin I, and $(M+H)^+$, at m/z 734.5, 772.4, 828.4, 837.4, 916.5, and 814.4 for erythromycin A, kitasamycin, josamycin, roxithromycin, tylosin A, and oleandomycin, respectively, were the precursor ions for collision-induced dissociation (CID) and 2 diagnostic product ions for each analyte were identified to carry out selected reaction monitoring (SRM) LC-MS/MS analyses. The recovery rates of macrolides in fish and shrimp samples were in the range 68.2-89.0, 72.8-91.6% and 75.2-87.0, 64.4-83.2% for the concentration of 50 and 100 ng/g, respectively.