Low serum vitamin D levels have been associated with a variety of health conditions which has led the medical community but also the general population to evaluate vitamin D status quite liberally. Nevertheless, there remain questions about the efficacy and cost-effectiveness of such a broad and untargeted approach. This review therefore aims to summarize the current evidence and recommendations on when and how to evaluate vitamin D status in human health and disease. For the general population, most guidelines do not recommend universal screening but suggest a targeted approach in populations at risk. Also, some guidelines do not even recommend evaluating vitamin D status when vitamin D substitution is indicated anyway, such as in children or patients receiving anti-osteoporosis drugs. In those guidelines that recommend the screening of vitamin D status, serum 25(OH)D levels are universally proposed as the preferred screening tool. However, little attention is given to analytical considerations and almost no guidelines discuss the timing and frequency of screening. Finally, there is the known variability in diagnostic thresholds for defining vitamin D insufficiency and deficiency. Overall, the existing guidelines on the evaluation of vitamin D status differ broadly in screening strategy and screening implementation, and none of these guidelines discusses alternative screening modes, for instance, the vitamin metabolic ratio. Efforts to harmonize these different guidelines are needed to enhance their efficacy and cost-effectiveness.