The aim of this study was to evaluate pain perception in epileptic children during an invasive procedure as the collection of venous blood through salivary alpha-amylase (sAA) activity determination, and to compare it with that of healthy children. In the study 23 children, 12 with epilepsy and 11 healthy controls were enrolled. From all children of both groups, one sample of saliva was collected through a non-invasive device, 15 min before (t 0), during (t 1), and 15 min later (t 2) blood withdrawal, and sAA activity was then determined through a kinetic-colorimetric assay. A statistically significant difference (p < 0.001) was found at t 2 between the sAA activity in the two groups, suggesting that epileptic children have an increased sensitization to pain, while at t 0 the difference was at the limit of statistical significance and at t 1 no statistically significant difference was found indicating that in both groups the venipuncture equally induced a state of stress. Our data suggest that sAA activity could represent a new objective and non-invasive biomarker for the assessment of pain perception in epileptic children.