Vitamin D deficiency has been linked to numerous health problems, including those resulting from disturbed calcium-phosphorus homeostasis, and neuropsychiatric and autoimmune disorders. Nearly one-third of the global population has suboptimal levels of vitamin D, according to epidemiological data. Vitamin D status is usually determined by measuring serum 25(OH)D, but, for decades, serum 25(OH)D measurement has been hampered by a lack of standardization. There have been many recent initiatives to develop reference substances and methods for measuring vitamin D and its metabolites, and re-evaluating the optimal values. It was also suggested that alternative biological samples could also be used, such as hair, since it has been established that lipophilic substances, such as corticosteroids, can also be found in hair. The purpose of this study was to determine the correlation between 25(OH)D3 concentrations in serum and hair, and other demographic features in 26 healthy Caucasian young adult volunteers. The determination of 25(OH)D3 and cholecalciferol was carried out using liquid chromatography coupled with mass spectrometry (LC-MS) from blood and hair samples taken at two timepoints separated by nine weeks. In the hair samples of 18 out of 26 subjects, 25(OH)D was detected at a mean (±SEM) concentration of 17.07 ± 5.375 pg/mg at the first sampling time, and 58.90 ± 25.97 pg/mg at the second sampling time. A multiple linear regression analysis revealed no effects of gender, body mass index, supplementation, or sun exposure on hair 25(OH)D3 concentrations, but supplementation and sun exposure significantly increased serum 25(OH)D3 concentrations. In addition, serum and hair 25(OH)D3 concentrations did not correlate; however, there was a strong correlation between the two sampling times for serum 25(OH)D3 concentrations. In conclusion, this study confirmed that 25(OH)D3 could be detected in human hair, but its use as a biomarker warrants further investigations since no link was found between serum 25(OH)D3 concentrations, supplementation, sun exposure, and hair 25(OH)D3 concentrations levels.