Vitamins are non-energy substances that are indispensable for the survival and development of organisms. All living processes associated with metabolism in the body are directly involved in the vitamin. In the present study, we determined optimal conditions to determine vitamin D2 content by ultra high performance liquid chromatography (UHPLC). The limit of detection (LOD) is 0.0064 μg/g, limit of quantification (LOQ) is 0.0195 μg/g. The precision and accuracy conforms to AOAC requirements. Thus, the method developed complies with international guides for the determination of vitamin D2 from higher fungi in North Central Vietnam. The results of vitamin D2 function analysis found in mushroom samples ranged from 6.221 - 11.755 μg/g.