This paper presents a liquid-chromatography negative ion electrospray tandem mass-spectrometry (LC-MS/MS) method to determine resorcylic acid lactones (RALs) in urine, muscle, fish, liver and kidney samples. The method comprises an extraction step followed by the clean-up of the samples in two steps using SPE cartridges: C18 Chromabond and Strata Amino from Phenomenex. The RALs were separated on a Pursuit C18 column. in isocratic mode with acetonitril/ 0.1% formic acid aqueous solution. The time for analysis was 12 minutes. A triple quadrupole mass spectrometric from Varian equipped with a ESI source in the multiple reaction monitoring was used for detection. The method was developed according to EU legislation and the parameters assessed were: selectivity, specificity, linearity, recovery, repeatability, within-laboratory reproducibility, decision limit (CC), and detection capability (CC), ruggedness, control chart, uncertainty of measurement. The validation was performed at 1μg/kg for each compound. CC and CC values were 0.16-0.79 micro g/kg and the range of mean recoveries were 72.2-119.7% depending of the compound. The relative standard deviation (RSD) of the measurements was below 20%.The method is used to analyze samples originating from the Romanian Residue Control Monitoring Program or private samples. The main purpose of this method is the detection, quantification and confirmation of 6 RAL’s in urine and tissue.