Individual monitoring of workers exposed to the risk of intake of actinides requires suitable methods for measuring low level of excreted activity. The current protocols used for actinides analysis in bioassay are usually complicated and highly time consuming. In this work, a protocol based on the microwave digestion of urines followed by the separation of actinides using calix[6]arene-based chromatography columns and their measurement by a quadrupole ICP-MS is developed and validated, for the first time, on urine samples containing the three actinides, U, Pu and Am. With this protocol, the total analysis time is about 2 days, including the mineralization of urine and the chromatographic separation of actinides. Detection limits of actinides in urine are determined and compared to those obtained after "dilute and shoot" ICP-MS analysis or after alpha spectrometry measurement.