Introduction: Our aim was to investigate biomarkers of neonatal pain and their association with two pain scales. Methods: This prospective study included 54 full-term neonates. Levels of substance P (SubP), neurokinin A (NKA), neuropeptide Y (NPY), and cortisol were recorded and two pain scales (Premature Infant Pain Profile [PIPP] and Neonatal Infant Pain Scale [NIPS]) were used. Results: A statistically significant decrease in the levels of NPY ( p = 0.02) and NKA ( p = 0.03) was detected. A significant increase in NIPS scale ( p < 0.001) and PIPP scale ( p < 0.001) postpainful intervention was also detected. There was a positive correlation between cortisol and SubP ( p = 0.01), NKA and NPY ( p < 0.001) and between NIPS and PIPP ( p < 0.001). A negative correlation was found for NPY with SubP ( p = 0.004), cortisol ( p = 0.02), NIPS ( p = 0.001) and PIPP ( p = 0.002). Conclusions: Novel biomarkers and pain scales may help in designing an objective tool for the quantification of neonatal pain in the everyday practice.