Oregano (Origanum spp.), a popular herb in western and Middle Eastern cuisine, was reported to show anti-inflammatory activities in vitro and in vivo but without any information as to the compounds responsible, whether the plants were authenticated or only contained true Origanum spp. Using a wide range of botanically authenticated oregano, we were able to show that oregano had anti-inflammatory activity and then using biodirected-guided fractionation, identified the anti-inflammatory agents in oregano as rosmarinic acid, oleanolic acid, and ursolic acid. In this study, we successfully developed an LC-MS (SIM mode) method to achieve coquantitation of these three organic acids with the application of a unique tandem column system. The detection of rosmarinic acid was optimal under negative ion mode of SIM, whereas oleanolic acid and ursolic acid were sensitive to positive ion mode. The simultaneous quantitation was attained by setting two time segments in one run to facilitate the ESI polarity switch. For the investigated analytes romarinic acid, oleanolic acid, and ursolic acid, good linearities (r(2) > 0.999) were obtained for each calibration curve. Validation for this method showed a precision (relative standard deviation) ranging from 4.84 to 6.41%, and the recoveries varied from 92.2 to 100.8% for the three analytes. A quantitative survey of these anti-inflammatory constituents in different oregano species (O. vulgare ssp. hirtum, O. vulgare, and O. syriacum) and chemotypes within the species varied significantly in their accumulation of rosmarinic, oleanolic, and ursolic acids. Significant variation in chemical composition between species and within a species was found.