A simple, rapid and highly sensitive method for the analysis of THC-COOH in urine, using automated on-line solid-phase extraction (SPE) combined with liquid chromatography (LC)-mass spectrometry (MS/MS), is developed and fully validated according to international guidelines. Chromatographic separation was achieved on an Atlantis dC(18) column with an isocratical gradient, ensuring the elution of THC-COOH within 4.1 min. The total process time was 6 min and 500 microL of sample was required. SPE using C(8) cartridges was highly effective, reproducible and led to significant decreases in the interferences present in the matrix. The method showed an excellent intra- and inter-assay precision (relative standard deviation (RSD) <7% and bias <13%) for four external quality control (QC) samples and three 'in house' QCs. Responses were linear over the investigated range (r(2)>0.99, 5-200 microg/L). Limits of quantification (LOQ) and detection (LOD) were determined to be 5 micro/L and 0.25 microg/L, respectively. Furthermore, the analyte and the processed samples were demonstrated to be stable. Moreover, no carryover was observed after the analysis of high concentrated urine samples (5000 microg/L THC-COOH)). The method was subsequently applied to authentic samples previously screened by a routine immunoassay method.