Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.