With an ever-increasing clinical interest in vitamin D insufficiency, numerous automated immunoassays, protein binding assays, and in-house LC-MS/MS methods are being developed for the quantification of 25-hydroxyvitamin D₃ (25(OH)D₃). Recently, LC-MS/MS methods have identified an epimeric form of 25(OH)D₃ that has been shown to contribute significantly to 25(OH)D₃ concentration, particularly in infant populations. This review describes the metabolic pathway and physiological functions of 3-epi-vitamin D, compares the capability of various 25(OH)D₃ methods to detect the epimer, and highlights recent publications quantifying 3-epi-25(OH)D₃ in infant, pediatric, and adult populations. In total, this review summarizes the information necessary for clinicians and laboratorians to decide whether or not to report/consider the C3-epimer in the analysis and clinical assessment of vitamin D status.