It has been proposed that developmental differences exist between neonatal and adult platelets. Detailed insight therein is, however, still lacking. We have now compared the platelet protein expression profile of neonates and adults employing a label-free quantitative mass spectrometry approach. In addition, platelet aggregation mediated by thromboxane A2 analog, collagen, and peptide agonists of the protease-activated-receptors 1 and 4 was assessed. Results showed that neonatal platelets effectively aggregate in the presence the employed platelet agonists. In agreement with previous studies, higher concentrations of the agonists were required to initiate aggregation in the neonatal platelets. Mass spectrometry analysis revealed no significant difference in the expression level of critical adhesive platelet proteins like glycoprotein (GP)Ib, integrin αIIbβ3, GPV and GPIX. Neonatal platelets did show reduced expression levels of proteins involved in intracellular signaling, i.e. LYN, MAP3k5 and FAM129A. Several proteins that are known to be related to mitochondrial energy metabolism processes such as oxidative phosphorylation, i.e. NDUFS3, NDUFS8 and NDUFA1 were upregulated in neonates. In conclusion, this study reveals that the platelets derived from neonates and adults are distinct. In particular, developmental changes were observed for proteins that belong to metabolic and energy generation processes.