Thirteen healthy men diagnosed with periodontitis were enrolled. Progression of periodontitis was monitored by clinical attachment level (CAL) changes at the site level over twelve weeks via weekly clinical evaluations and gingival crevicular fluid (GCF) samples were collected using cellulose strips for subsequent mass spectrometry analysis. Progression group (PG) included 18 GCF samples matching sites with disease progression (CAL ≥ 2 mm), while the non-Progression group (NP) included 18 GCF samples from stable sites (No CAL changes) matched in the same patients. Clinical metrics, HPRLC-MS/MS, in silico methods, and bioinformatics tools, were employed to analyze the proteome of GCF, revealing the significant occurrence of ferroptosis during clinical progression of periodontitis.