Determination of folate (FA) or vitamin B9 and its derivatives such as 5-methyltetrahydrofolate (5-MTHF), Tetrahydrofolate (THF) and 10-formyl folic acid (10-CHOFA) in Thai rice with reverse-phase high-performance liquid chromatography. This technique was a simple and fast method for the simultaneous determination of folate and its derivatives. The seed of rice samples were collected from the Department of Genetics, Faculty of Science, Maejo University. Rice powder (0.5000 g) was soaked in 1 mL water and shaken at room temperature and centrifuged at 3500 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm Nylon membrane filter and 20 μL was injected into the HPLC. Separation was performed using a C18 column with isocratic elution of 0.1% formic acid: acetonitrile (85:15 v/v). The flow rate was 0.70 mL min-1 and a photodiode array detector at the wavelength of 267 nm was employed. The calibration curve obtained (R2 > 0.9990) for folate, 5-MTHF, THF and 10-CHOFA were in the range of 0.50-50.0, 0.50-200.0, 0.50-50.0 and 5.0-100.0 mg L-1, respectively. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.09- 0.15 mg L-1 and 0.30 to 0.50 mg L-1. Percent recovery at the concentrations of 15 and 35 mg L-1 were found in the range of 95-113. In the ten studied samples, folate and its derivatives were found in the range of 0.19-27.10 mg (in 100 g dry weight). This technique was applied to simultaneous determination of folate and derivatives in rice.