Analysis of antibiotics in the environment has become an urgent issue. A novel method entailing microwave-assisted extraction (MAE) and high-performance liquid chromatography–electrospray mass spectrometry (LC–MS n ) has been developed for determination of selected typical antibiotics, including quinolones, sulfonamides, and tetracyclines, in manure. Compared with ultrasonic extraction, MAE significantly increased recovery of fluoroquinolones (63–106%), sulfonamides (64–133%), and tetracyclines (64–109%) from manure. Acetonitrile acidified with formic acid buffer solution (pH 4.0) was used for extraction of swine manure whereas chicken manure was extracted with 0.1 M EDTA–McIlvaine buffer solution. Limits of quantification (LOQ) for all compounds were in the range 5.12–168.4 μg kg−1 dry matter, which were satisfactory for analysis of all samples. The suitability of the method was assessed by analysis of manure from six different sites.