Abstract: A sensitive and reliable multi-residue method has been developed for the simultaneous determination of six resorcylic acid lactones in fat. The samples were extracted with acetonitrile followed by solid-phase extraction using NH₂ cartridges. The compounds of interest were detected by multiple reaction monitoring using electrospray ionization negative mode. Mean recoveries from fortified samples ranged from 80.1% to 107.1%, with coefficients of variation lower than 10.7%. The limits of detection (LOD) and quantitation (LOQ) for resorcylic acid lactones were 0.5 μg•kg^－1 and 1.0 μg•kg^－1, respectively.