Newborns are exposed to a considerable number of painful stimuli. This study is aimed to investigate the effects of 30% glucose solution and nonnutritive sucking on pain perception during venipuncture. Twenty-five term infants were randomized as receiving 30% dextrose (group 1) or sterile water (group 2). Neonatal Infant Pain Scale scores, skin conductance algesimeter recordings, and near-infrared spectroscopy measurements were recorded during the procedure. Neonatal Infant Pain Scale and skin conductance algesimeter results were decreased in both groups from that during venipuncture to after the procedure. Group 1 had lower Neonatal Infant Pain Scale scores compared with group 2 after venipuncture, different from the skin conductance algesimeter, where no difference was observed between groups. In group 1, cerebral blood volume increased after venipuncture. Glucose does not attenuate the Neonatal Infant Pain Scale score and skin conductance algesimeter index during venipuncture, but it leads to a lower Neonatal Infant Pain Scale score after venipuncture unlike the skin conductance algesimeter index, which was not lowered.