Two methods are suggested for determination of panthenol. The first is colorimetric method where panthenol is subjected to alkaline hydrolysis; the resulting β-alanol is allowed to react with vanillin (Duquenois reagent) in presence of McIlvain buffer pH 7.5. The color developed is measured at 406 nm. The linearity range was found to be 50-500 μg/ml while the lower limit of detection was about 10 μg/ml. The second is a sensitive and reliable modified fluorimetric method is also suggested, whereas panthenol, after alkaline hydrolysis is treated with ninhydrin. The fluorescent product was found to have excitation λ m a x at 385 nm and emission λ m a x at 465 nm. The method showed high sensitivity with linearity range from 0.01 to 3 μg/ml. The lower limit of detection (LOQ) reached 0.005 μg/ml. Validation of both methods was carried out and the two methods were applied for determination of panthenol in some cosmetic and pharmaceutical formulations.