Vitamin D deficiency and insufficiency has become a pandemic health problem with a consequent increase of requests for determining circulating levels of 25-hydroxyvitamin D [25(OH)D]. However, the analytical performance of these immunoassays, including radioimmunoassay and ELISA, is highly variable, and even mass spectrometric methods, which nowadays serves as the gold standard for the quantitatively determination of 25(OH)D, do not necessarily produce comparable results, creating limitations for the definition of normal vitamin D status ranges. To solve this problem, great efforts have been made to promote standardization of laboratory assays, which is important to achieve comparable results across different methods and manufacturers. In this review, we performed a systematic analysis evaluating critically the advantages and limits of the current assays available for the measure of vitamin D status, i.e., circulating 25(OH)D and its metabolites, making suggestions that could be used in the clinical practice. Moreover, we also suggest the use of alternatives to blood test, including standardized surveys that may be of value in alerting health-care professionals about the vitamin D status of their patients.