Mushrooms are known to contain an array of nutrients such as amino acids, lipids and carbohydrates and important neutraceuticals including tocopherols, phenolics, flavonoids, carotenoids, vitamins and other health-promoting constituents.   For the past decade there has been an increase interest in mushrooms as a potential source of vitamin D.  It has been shown that irradiation of the Agaricus bisporus species with UV converts the sterol, ergosterol to vitamin D2 (ergocalciferol).  This treatment significantly increases the vitamin D content up to levels needed for a daily adult intake.  UV irradiation of mushrooms is therefore used to provide a dietary source rich in vitamin D especially in regions where vitamin D deficiency is prevalent.  However, little is known about how exposure to UV affects other molecules in the fungi.  We adopt a metabolomics based approach to profile untreated mushrooms using two-dimensional liquid chromatography (2DLC) and further investigate the changes in metabolite content.  An offline reversed-phase 2DLC method has been developed in order to achieve maximum peak capacity and resolve the components of this complex sample.  This methodology is applied to Agaricus bisporus mushrooms treated in various UV conditions to offer a better understanding of the treatment and its effect on the beneficial biochemicals in particular.