Maslinic acid is a bioactive compound found in olives and olive oil to which a myriad of beneficial effects have been attributed, such as antioxidant, antitumor and antidiabetic. Here, we present a high performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method to determine maslinic acid and its metabolites in rat intestinal content after the oral administration. The pentacyclic triterpene was orally administered to three groups of male Sprague-Dawley rats at single daily doses of 5, 2 and 1 mg/kg, respectively. At 24 h after the last administration, the content of the duodenum and jejunum, ileum, caecum and colon was obtained. Analytes were extracted with methanol 80% by homogenization and stirring. Chromatographic separation was carried out using a gradient elution of water and acetonitrile and detection was performed in a single quadrupole mass spectrometer, with an atmospheric pressure chemical ionization (APCI) source. The metabolites were identified by LC-APCI-LTQ-Orbitrap-MS. The analytical method was validated by spiking blank intestinal content samples with maslinic acid at the concentrations of 0.1, 0.5, 1, 5, 10, 20 and 30 µM. The mean coefficient of correlation was 0.994, the mean recovery was 97.87 ± 0.76% and the limit of quantification was 5 nM. The intra- and inter-day precisions were 4.32 ± 1.69% and 6.22 ± 1.05%, respectively. After 24 h of the last administration of maslinic acid, the analysis of the content of the different intestinal segments demonstrated the presence of maslinic acid (C30H48O4) together with nine metabolites: five monohydroxylated derivatives (C30H48O5) (M1-M5), two monohydroxylated and dehydrogenated metabolites (C30H46O5) (M6-M7) and two dihydroxylated derivatives (C30H48O6) (M8-M9). Metabolites of the same m/z differed by the retention time (t). The percentage that each compound represented over the total amount along the small and large intestine was calculated. Maslinic acid accounted for 18.9 ± 1.2% and the major metabolites were the monohydroxylated M1(t = 6.6 min), representing an average of 55.4 ± 2.3%, followed by M2 (t = 7.3 min), which accounted for 17.0 ± 5.8%. To a lesser extent, the monohydroxylated and dehydrogenated metabolites M6 (t = 6.8 min) and M7 (t = 8.1 min) represented an average of 4.9 ± 2.8% and 2.8 ± 0.7%, respectively, and the rest of the derivatives (M3, M4, M5, M8 and M9) accounted for less than 1%. These results show that 24 h after the oral administration, maslinic acid undergoes extensive phase I metabolism, following the same pattern at three different doses. Two monohydroxylated derivatives are the most abundant, one of them accounting for more than double of the maslinic acid fraction in the whole intestinal content.