<b><i>Background:</i></b> Oral food challenge (OFC) is commonly used to diagnose food allergy. This test is time and resource intensive, and conclusions are not always unequivocal as this relies on the interpretation of symptoms. Therefore, an objective marker would improve the accuracy of the diagnostic workup of food allergy. <b><i>Objectives:</i></b> The aim of this study was to investigate whether tryptase can be detected in saliva of children following OFC. <b><i>Method:</i></b> Children from 3 to 18 years of age were eligible for inclusion if an OFC for peanut or tree nut had been recommended. Saliva samples were collected prior to the first dose and 5, 10, and 15 min following the last administered dose during OFC. Assay precision, spike-and-recovery, and assessment of lower limit of detection of the tryptase immunoassay were examined before analysis of tryptase in saliva was performed. <b><i>Results:</i></b> A total of 30 children were included (median age 8 years, 63.3% male, 53.3% positive OFC outcome). Tryptase was detected in saliva samples. The mean of the change in baseline tryptase value to each saliva collecting time point was significantly different in patients with a positive OFC outcome compared to a negative outcome (<i>p</i> &#x3c; 0.01). <b><i>Conclusions:</i></b> This study showed that tryptase can be detected in saliva of children following OFC. Increased levels of tryptase compared to baseline were found if the OFC outcome was positive, suggesting that measuring tryptase in saliva may be useful in the diagnosis of food allergy. Further research is needed to evaluate the potential association between tryptase levels and symptoms.