Purpose: A simple and high-throughput analytical method for determining clitidine in Paralepistopsis acromelalga using hydrophilic interaction liquid chromatography–tandem mass spectrometry (LC–MS/MS) was established. Methods: To determine clitidine in the mushrooms, a simple procedure including dilution with methanol solution and filtering by cartridge was employed just before quantification by LC–MS/MS for high-throughput analysis. In this report, concentrations of clitidine in mushrooms were determined by the standard addition method. Results: The present established method was successfully applied to the analysis of fruit bodies of P. acromelalga, which were obtained from five different locations in Japan. Results on concentrations of clitidine in each stem and cap of P. acromelalga specimens tested showed that their concentrations were quite different, not only between stems and caps, but also among locations and strains; the concentrations of clitidine in stems and caps ranged from 1.41 to 9.30 mg/g and 3.17 to 14.4 mg/g, respectively. Conclusions: This is the first report to present a detailed quantitative analysis of clitidine by MS and the distribution of clitidine in stems and caps of P. acromelalga. This analytical method for clitidine was thought to be useful in P. acromelalga poisoning cases to identify the causative toxic mushroom.