Mast cell activation syndrome (MCAS) describes patients with episodes of mast cell mediator release, with negative bone marrow biopsy results, and the failure to meet the criteria for systemic mastocytosis.Identify elevation of mast cell mediators of patients with MCAS.We performed a retrospective study of 25 patients with MCAS who were evaluated at Mayo Clinic from 2006 to 2012. Patients were reviewed for MCAS symptoms and mast cell mediators, including serum tryptase and 24-hour urine N-methyl histamine (N-MH) and 11β-prostaglandin-F₂α (11β-PGF₂α). The study was approved by the institutional review board.Urinary 11β-PGF₂α was the most frequently elevated product in MCAS of our 25-patient cohort. Flushing and pruritus had the greatest correlation with elevation of 24-hour urine 11β-PGF₂α value at baseline. The serum tryptase level was elevated in 10 patients, whereas the N-MH level was elevated with 2 patients. Eight of 9 patients with MCAS and with elevated 24-hour urine 11β-PGF₂α who underwent aspirin therapy and follow-up urinary studies had normalization of this mediator (1 patient did not have a follow-up urine study). Six of these 9 patients with MCAS who underwent aspirin therapy had symptomatic improvement.We recommend measurement of 24-hour urine 11β-PGF₂α and serum tryptase levels of patients with symptoms suggestive of MCAS. Measurement of 24-hour urine 11β-PGF₂α and serum tryptase levels can help avoid misdiagnosis and overinterpretation of MCAS symptoms in clinical practice. Given that an elevation of 24-hour urine N-MH level was found only in 2 patients, measurement of this mediator may be less helpful in diagnosing MCAS. We recommend aspirin therapy for patients with MCAS and with elevated 24-hour urine 11β-PGF₂α levels.