Periodontitis is characterized by gingival regression, alveolar bone resorption and the development of deep periodontal pockets that, if left untreated, can lead to tooth loss. Currently, specific biomarkers are needed for the early, objective diagnosis, monitoring, and management of periodontal patients. In this proteomic study, periodontal pocket tissues from patients with severe periodontitis were analyzed in comparison to periodontally healthy sites with the aim of discovering distinctive protein targets. Gingival tissues were fragmented using a motorized mechanical method and mixture protein was separated via mono-dimensional gel electrophoresis. The examination of protein bands using definite 1D image analysis software allowed for the detection of 22 differentially expressed proteins between pathological and healthy samples that were identified through mass spectrometry. A comparative assessment of these proteins with those previously reported in other studies conducted on periodontal diseases in various types of oral specimens, such as gingival crevicular fluid, dentin, tooth pulp, root canal content, salivary gland secretions, saliva, periodontal ligament cells, and dental stem cells, highlighted a great number of significant common matches. The discovery of a selective cluster of periodontitis-related biomarkers could become particularly important before the clinical manifestation of the disease to promptly stop its progression for a timely preventive diagnosis.