Background: Serum total tryptase has been shown to increase during acute allergic reactions (acute tryptase, TA ); however, few studies have investigated the values of TA or a combination of TA and baseline tryptase (TB ) to discriminate positive from negative testing in perioperative hypersensitivity reaction (POH) allergy work-up. The aim of this study was to determine the diagnostic performance of TA in order to differentiate positive from negative allergy testing suspected POH and analyse the diagnostic performance of serial tryptase levels using several formulas. Methods: All patients from the University hospital of Montpellier and Strasbourg, France, who presented with suspected POH and underwent complete drug allergy work-up between March 2011 and December 2019 with available TA and TB were included. Four formulas, including a change in TA > 11 (F1), or >2 + 1.2 × TB (F2), or >3 + TB (F3), or >120%TB (F4), were applied. Results: One hundred and sixty-two patients were included, and 131 of them (80.8%) had Grade III or IV reactions. Ninety patients had positive allergy testing. The optimal cut-off value of TA to distinguish positive from negative allergy testing patients was 9.8 μg/L with an AUC of 0.817 (95% CI: 0.752-0.882, p < .001). The 93% PPV threshold for TA was 33 μg/L (95.8% specificity). Paired tryptase levels according to formulas F2 and F3 yielded the highest Youden index (0.54 and 0.53, respectively). Conclusion: The optimal cut-off point for TA for distinguishing positive from negative allergy testing suspected POH was 9.8 μg/L. TA value of 33 μg/L was required to achieve >90% PPV.