This paper describes a rapid and robust method utilizing microwave-assisted derivatization, automated SPE, and LC-MS/MS for the quantitation and confirmation of chloramphenicol (CAP) and nitrofuran metabolites in various aquaculture matrixes. The use of equipment presented in this work allowed extractions to be completed on average within 6 h, with quantitation accuracy ranging from 89 to 107% and RSD≤8.3%. The demonstrated detection limits for all the nitrofuran metabolites of interest in three different matrixes were ≤0.06 ng/g, with a quantitation limit of ≤0.2 ng/g. Additionally, the method exhibited a CAP detection limit for all matrixes≤0.01 ng/g and an LOQ of ≤0.03 ng/g.