A selective reversed-phase high-performance liquid chromatography (RP-HPLC) technique was developed for 1α-hydroxy vitamin D2 (Doxercalciferol) related substances analysis. The technique is proficient of eluting various impurities related to process and degradation. To separate both the vitamin D2 Analogs i.e., doxercalciferol and ergocalciferol. HPLC of Shimadzu, provided with autosampler, column oven, quaternary pump and UV detector, were used for development studies. The mobile phase used water and acetonitrile. The diluent used was methanol and detection was examined at wavelength 265 nm. The method is sensitive for doxercalciferol, ergocalciferol, impurity-A and impurity-B; the limit of detection (LoD), value is 0.01% and limit of quantitation (LoQ) value is 0.03%. Linearity of doxercalciferol and related impurities at various concentrations was performed and the obtained correlation coefficient for doxercalciferol is 0.995, ergocalciferol is 0.997, impurity-A is 0.990 and impurity-B is 0.993. The optimized method was validated and demonstrated that the technique is selective, precise, linear, sensitive and accurate. The method is simple, inexpensive and useful for routine testing of doxercalciferol API. In addition, the technique was effectively applied for the elution of both the vitamin D2 Analogs i.e., doxercalciferol and ergocalciferol. Further, the same method is applicable for the determination of assay for these two API’s and the same assay method was validated.