Previous studies have assessed vitamin D status based on the 25-hydroxyvitamin D [25(OH)D] concentration measured in samples from dried blood spots (DBSs). In 40 individuals participating in a clinical study, we compared 25(OH)D levels measured from DBSs and in serum using an LC-MS/MS reference procedure in collaboration with the Vitamin D Standardization Program. The main objective was to simplify and optimize current methods to produce an assay that can be used as a screening tool for 25(OH)D concentration assessment without derivatization. The DBS 25(OH)D levels, compared to serum concentrations, were found to have 101% accuracy overall, and the correlation coefficient (r) was 0.83 (P < 0.0001), with a significant linear relationship. Free 25(OH)D and vitamin D binding protein (VDBP) were assessed in the serum samples for potential correlations to the DBS calculations: the levels of free 25(OH)D had moderate to strong correlation to DBS and serum concentrations, with r values of 0.67 (P < 0.0001) and 0.76 (P < 0.0001), respectively. VDBP and hematocrit had no significant correlation to either DBS or serum sample types, with r values <0.1. In conclusion, the use of two DBSs and an increase in DBS sample size improved overall sample representation without the need for derivatization, and produced an accurate and robust method that can be used to screen 25(OH)D levels.