A simple and economical method has been developed for simultaneous determination of human serum 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) using Ultra Performance Liquid Chromatography (UPLC). Non-human matrix of 4% BSA was used to construct the calibration curve and in quality control samples' preparation to avoid interference of the endogenous 25-hydroxyvitamin D (25OHD) present in the human serum. 25OHD2, 25OHD3 and dodecanophenone (internal standard, IS) were separated on a CORTECS solid-core particle column and monitored by photodiode array detector at wavelength of 265 nm within five min run time. The relationship between 25OHD concentration and peak area ratio (25OHD:IS) was linear over the range of 12.5 - 200 nM with mean correlation coefficients (r2) >0.998. The limit of detection (LOD) for 25OHD2 and 25OHD3 was 3.00 nM and 3.79 nM, while the lower limit of quantification (LLOQ) was 9.11 nM and 11.48 nM, respectively. High repeatability was obtained for both isomers with intra-day CV% <5.6% and <5.3% for inter-day assay. This method was further tested with a commercial lyophilized serum control with an accuracy of 92.87-108.31% and applied on 214 human serum samples. In summary, this validated method with BSA can be reliably applied for routine quantification of 25OHD in adults.