Ractopamine glucuronides have been identified in cattle urine sampled by LC-MS/MS. An ELISA method, which was capable of specifically determining (1R, 3R)-ractopamine stereoisomer and its glucuronide metabolites, had more than 100% recovery with an acceptable coefficient of variation in the inter- and intra-assay variation tests for RR-ractopamine. The concentration levels of parent ractopamine and ractopamine glucuronide metabolites as the main components of total ractopamine in cattle and sheep urine showed similar depletion trends, in which the concentration curves increased and reached a climax during the feeding period, and then dropped quickly when entering the withdrawal period. Data from the three methods had very good pair-wise correlations. In the cattle urine samples, the correlation coefficient (R(2)) for parent ractopamine between the ELISA and the LC-MS/MS or GC-MS results were 0.93 or 0.92; R(2) values for parent ractopamine and total ractopamine data measured by LC-MS/MS and GC-MS were 0.9651 and 0.9677, respectively. All R(2) values for data gained from sheep urine samples were >0.95. The study indicated that the close levels of RR-ractopamine stereoisomer in cattle and sheep urine samples may imply the presence of a similar depletion pattern in other livestock, and thus would facilitate an accurate detection and management of ractopamine usage in food safety.