Vitamin D, also known as the sunshine vitamin, is both a micronutrient and a prohormone. There are two structurally different forms of vitamin D: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Both forms can be taken into the body through food or dietary supplements. In addition, ergocalciferol and cholecalciferol can be synthesized non-enzymatically by UVB radiation of sunlight from provitamin forms, which are ergosterol and 7-dehydrocholesterol. In this study, a methodology based on molecularly imprinted solid phase extraction (MISPE) is intended prior to the determination of D2 and D3 by HPLCDAD. In accordance with this purpose, precipitation and sol-gel polymerization methods were used to synthesize molecularly imprinted polymers (MIPs) and nonimprinted polymers (NIPs). Also, different templates, monomers, crosslinkers, and their ratios were tried in the synthesis of MIPs/NIPs by precipitation polymerization. Among all syntheses, MIP, which was synthesized using vitamin D2 as template (T), 4- vinylpyridine (4-VP) as monomer (M), ethylene glycol dimethacrylate (EGDMA) as crosslinker (C) at a molar ratio of 1:6:30 (T:M:C), enabled the co-determination of vitamin D2 and D3. It selectively worked on the vitamin D prohormone group compared to both NIP and commercial solid phase extraction (SPE) sorbents in the presence of other structurally related compounds. The critical experimental parameters of the MISPE method were optimized for the analyte concentration of 1.0 mgL-1 and determined as follows: 8 hours as the sorption time, 5.0 mg as the sorbent amount, 5.0 mL as the sample solution volume, methanol as the eluent, and 24 hours as the desorption time. A matrix-matched calibration curve with optimized parameters was proposed. The applicability of the developed MISPE method was confirmed by analyzing the sample of vitamin D3 supplementation and overall recovery was found as 95.97% (±1.84) for n=2.