Background: British guidelines recommend that serial acute serum tryptase measurements be checked in all adults and a subset of children presenting with anaphylaxis. This is the first study reporting the clinical utility of acute serum tryptase in a “real-world” emergency department (ED) setting following the publication of the World Allergy Organization (WAO) criteria for anaphylaxis. Objectives: To (1) assess sensitivity, specificity, and positive and negative predictive values (PPV, NPV) of acute serum tryptase in anaphylaxis; (b) determine factors associated with higher acute serum tryptase levels; and (c) audit compliance of acute serum tryptase measurement in the ED. Methods: The methods used were retrospective electronic search for ED admissions to 3 acute care hospitals in Birmingham, UK, with anaphylaxis in 2012 using wide search terms followed by scrutiny of electronic clinical records and application of the WAO diagnostic criteria for anaphylaxis. Patients with an acute serum tryptase measurement were included in the analysis. Results: Acute serum tryptase level was measured in 141 of 426 (33.1%) cases. Mean time from the onset of symptoms to the measurement of acute serum tryptase level was 4 hours 42 minutes (SD ± 05:03 hours) and no patients had serial measurements conforming to British guidelines. Acute serum tryptase level of more than 12.4 ng/mL (75th centile) was associated with a sensitivity, specificity, PPV, and NPV of 28%, 88%, 0.93, and 0.17, respectively. Multiple regression analysis showed that male sex (odds ratio, 2.66; P = .003) and hypotension (odds ratio, 7.08; P = .001) predicted higher acute serum tryptase level. Conclusions: An acute serum tryptase level of more than 12.4 ng/mL in an ED setting carries high PPV and specificity, but poor sensitivity and NPV