Determination of asperulosidic acid (AA) and deacetylasperulosidic acid (DAA) in rat plasma after administration of Morinda citrifolia juice by HPLC-UV detection was described. Deproteinized rat plasma (200 L) with MeOH for the HPLC analysis was injected. AA and DAA was separated on ZIC^®-HILIC (250×4.6 mm, i.d., 5 μm) with a mixture of CH₃CN/MeOH/0.1% formic acid aqueous solution (=70:25:5, v/v/v) at a flow rate of 0.75 mL/min. The absorbance of eluate was monitored at 235 nm. Under this condition, the separation of AA and DAA was achieved within 25 min. The calibration curves using plasma spiked with standards indicated good linearity (r≥0.996) in the range of 0.8-20 g/mL for AA and 4-100 μg/mL for DAA, respectively. The limits of quantitation for AA and DAA at a signal-to-noise ratio of 10 were 0.3 and 1.1 μg/mL, respectively. The validation parameters of the method such as recovery (94.9-101.7 %), precisions (less than 7.1 % for intra-day and less than 9.8 % for inter-day) and accuracy (95.0-101.7 %) were acceptable. The analytes in rat plasma were stable after three freeze-thaw cycles or storage at room temperature for up to 6 h. Furthermore, monitoring of AA and DAA after administration of the Morinda citrifolia juice in which AA and DAA were determined, was successfully demonstrated. This is the first report to determine AA and DAA in rat plasma after administration of Morinda citrifolia juice.