Background: There are questions about the appropriate method for the accurate estimation of the population prevalence of nutrient inadequacy on the basis of a biomarker of nutrient status (BNS). Objective: We determined the applicability of a statistical probability method to a BNS, specifically serum 25-hydroxyvitamin D [25(OH)D]. The ability to meet required statistical assumptions was the central focus. Design: Data on serum 25(OH)D concentrations in adults aged 19–70 y from the 2005–2006 NHANES were used (n = 3871). An Institute of Medicine report provided reference values. We analyzed key assumptions of symmetry, differences in variance, and the independence of distributions. We also corrected observed distributions for within-person variability (WPV). Estimates of vitamin D inadequacy were determined. Results:We showed that the BNS [serum 25(OH)D] met the criteria to use the method for the estimation of the prevalence of inadequacy. The difference between observations corrected compared with uncorrected for WPV was small for serum 25(OH)D but, nonetheless, showed enhanced accuracy because of correction. The method estimated a 19% prevalence of inadequacy in this sample, whereas misclassification inherent in the use of the more traditional 97.5th percentile high-end cutoff inflated the prevalence of inadequacy (36%). Conclusions: When the prevalence of nutrient inadequacy for a population is estimated by using serum 25(OH)D as an example of a BNS, a statistical probability method is appropriate and more accurate in comparison with a high-end cutoff. Contrary to a common misunderstanding, the method does not overlook segments of the population. The accuracy of population estimates of inadequacy is enhanced by the correction of observed measures for WPV.