Folate characterization has remained an important, yet complex problem dating from the initial discovery (ca. 1931) by Lucy Wills that yeast contained an active component capable of curing macrocytic anemia in pregnant women. Since that time, qualitative and quantitative analysis of folates has been hindered by their inherent instability, multiplicity of forms and minute levels in nature. Folate deficiency is recognized as a serious health issue in many parts of the world, and most recently, folate deficiency has been associated with increased risk of developing cardiovascular disease. New, more powerful analytical approaches, such as those based on mass spectrometry (MS), are currently being applied to folate characterization. Fast atom bombardment (FAB), electron-impact (EI), plasma desorption (PD) and secondary-ion MS techniques have been used for structural characterization of folates. Hyphenated techniques based on either liquid chromatography (LC/MS or LC/MS/MS) or gas chromatography (GC/MS), as well as matrix-assisted laser-desorption time-offlight MS (MALDI-ToF MS) and accelerator MS (AMS) have been used for folate quantitation. A review of the past and current MS-based approaches to folate analysis in relation to structural, nutritional and clinical research is described in the present report. Recommendations for future MS-based applications are also presented.