In the last decade the scientific and medical community was confronted with a renewed interest in vitamin D and its metabolites, interest prompted by new discoveries regarding the association between members of the vitamin D family and a great number of physiological functions and pathological states. An impressive number of research projects have helped clear the path towards a better understanding of the functions of vitamin D and have resulted in the development of numerous methods of analysis. This review focuses on the various extraction methods used for analysis of vitamin D in research or clinical settings. Two main extractive methods are usually employed: liquid-liquid extraction and solid-phase extraction. Some methods use no extraction step and direct analysis is performed at the cost of significantly increased matrix interference. On the other hand, other methods use combined extraction techniques, and even additional derivatization steps in order to increase the sensitivity and accuracy of the analysis. The method of choice ultimately depends on the research question and the purpose of the study.