The performances of four LC-MS/MS methodologies for determination of up to eight mycotoxin biomarkers in human urines were compared by involving three laboratories that analysed common urine samples spiked at two levels of each biomarker. Each laboratory received a calibration solution, spiked urines and the corresponding unspiked urine. The two spiking levels for each biomarker were chosen by considering the levels naturally occurring in human urines and the limits of quantification of the LC-MS/MS methodologies used by the participating laboratories. The results of each laboratory were evaluated for their z-score values. The percentage of satisfactory z-scores (| z | < 2) were: 100% for deoxynivalenol, de-epoxy deoxynivalenol, aflatoxin M1, β-zearalenol and zearalenone, 87% for α-zearalenol, 50% for ochratoxin A and 42% for fumonisin B1. Good method performances were obtained for most biomarkers at the levels tested in this study, as demonstrated by the overall percentage of satisfactory z-scores for all analytes (87%). Unsatisfactory/questionable z-scores (| z | ≯2) were obtained for fumonisin B1 (7/12 results), ochratoxin A (4/8 results) and ?-zearalenol (1/8 results). The percentage of satisfactory z-scores for fumonisin B1 and ochratoxin A increased from 42 to 83% for fumonisin B1 and from 50 to 62% for ochratoxin A when laboratories 1 and 2 used own calibrants. Factors that could explain the different results obtained for fumonisin B1 and ochratoxin A with provided and own calibration solutions could not be identified in this study and should be carefully investigated in future studies.