Gingival Crevicular Fluid, a plasma-derived exudate present in the gingival crevice was collected from deciduous, exfoliating and permanent teeth from 20 children (60 samples) with the aim to characterize and quantify by a mass spectrometry based top-down proteomic approach, the peptide/proteins in the fluid and verify possible variations occurring during the exfoliating process. The results obtained confirmed the presence in Gingival Crevicular Fluid of α-Defensins 1-4, Thymosin β4 and Thymosin β10, as described in previous works and revealed the presence of other interesting peptides never described before in Gingival Crevicular Fluid, such as specific fragments of α-1-antitrypsin, α-1-antichymotrypsin, Thymosin β4 and Thymosin β10 fragments, Fibrinopeptide A, Fibrinopeptide B, S100A, LVV Hemorphin-7 (hemoglobin chain β fragment), as well as some other peptides deriving from α and β subunits of hemoglobin. Statistical analysis evidenced different levels in 5 proteins/peptides in the three groups in particular with higher level in exfoliating teeth. Our study demonstrate that an in-depth analysis of a biological fluid like Gingival Crevicular Fluid, present in small amount, can provide useful information for the understanding of different biological processes like teeth eruption.