Direct determination of urinary mycotoxins is a better approach to assess individual's exposure than the indirect estimation from average dietary intakes. In this study, a new analytical method was developed and validated for simultaneous analysis of aflatoxin B₁, deoxynivalenol, fumonisin B₁, ochratoxin A, zearalenone and T2 toxin and their metabolites in pig urine. In total 12 analytes were selected. A salting-out assisted liquid–liquid extraction procedure was used for sample preparation. High performance liquid chromatography/tandem mass spectrometry was used for the separation and detection of all the analytes. The extraction recoveries were in a range of 70–108%, with the intra-day relative standard deviation and inter-day relative standard deviation lower than 25% for most of the compounds at 3 different concentration levels. Meanwhile the method bias for all the analytes did not exceed 20%. The limits of quantification ranged from 0.07ngmL⁻¹ for ochratoxin A to 3.3ngmL⁻¹ for deoxynivalenol. Matrix effect was evaluated in this study and matrix-matched calibration was used for quantification. The developed method was also validated for human urine as an extension of its application. Finally, the developed method was applied in a pilot study to analyze 28 pig urine samples. Deoxynivalenol, aflatoxin B₁, fumonisin B₁ and ochratoxin A were detected in these samples.