Methotrexate (MTX) is widely used as antineoplastic drug (AD) and as an immunosuppressive. As a result, many healthcare professionals are exposed to this drug which is classified as dangerous to handle due to its reproductive toxicity in humans. Since the 1990 s, cases of internal contamination of professionals handling this molecule have been reported in the literature and even recently MTX was detected in the urine of professionals. To date, there is no toxicological reference value for occupational exposure to MTX. Given the toxicity of this molecule, the internal contamination of professionals must be reduced and kept as low as possible according to the ALARA principle (as low as reasonably achievable). The aim of this work was to develop an UHPLC-MS/MS method in MRM (Multiple Reaction Monitoring) and MRM(3) modes for routine application in MTX occupational biomonitoring. Good linearity (r greater than 0.997), precision (CV < 15 %), and accuracy (94.97-97.80% of the nominal value in MRM mode; 105.90-112.25% in MRM(3) mode) were achieved. This method is reliable with high specificity and high sensitivity especially in MRM(3) mode and has better LOD and LLOQ (1 ng/L and 2.5 ng/L) than published methods to date. The MRM(3) mode increases the signal-to-noise ratio compared to the MRM mode. It was then applied routinely for the biological monitoring of healthcare professionals exposed to methotrexate. One hundred and seventeen urine samples from 93 healthcare professionals occupationally exposed to methotrexate were analyzed. Fifteen healthcare professionals (16.1 %) were found to be contaminated with methotrexate. Urine concentration levels ranged from 2.5 to 380 ng/L with a median value of 8.9 ng/L. Such efficient analytical tool is essential for the routine biological monitoring of healthcare professionals exposed to methotrexate. It also enables the traceability of occupational exposure to this molecule and the evaluation of the effectiveness of preventive measures such as individual and collective protective equipment.< Réduire