In recent years, dietary intake of vitamin D has become an issue of high concern because this bioactive molecule boosts the immune system and is presumed to provide some protection against Covid-19. Under these conditions, a search for nontraditional dietary sources has appeared as a new challenge. One of the possibilities is irradiation of champignons that contain a high amount of ergosterol, a vitamin D2 precursor. In our study, a fast and sensitive liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) method for the determination of vitamin D2 in fresh mushrooms and its metabolite 25(OH)D2 in the blood of volunteers regularly consuming UV-treated mushrooms has been introduced. For extraction of desiccated mushrooms, solid-liquid extraction n-hexane–ethyl acetate was used, and n-hexane was employed for blood plasma samples. Separation of target analytes was performed on a polymeric bonding C18 phase column. Satisfactory limits of quantification (LOQs) were reached both for the control of vitamin D2 content in mushrooms (LOQ = 10 ng/g) and for the monitoring of vitamin D2 and D3 metabolite in human blood (LOQ = 2.5 ng/mL). For accurate quantification, isotopic dilution was employed.