CIGB-814, originally named as E18-3 APL1 or APL1 in preclinical experiments, is a novel therapeutic peptide candidate for Rheumatoid Arthritis (RA). It is an altered peptide ligand containing a novel CD4+ T-cell epitope of human heat shock protein 60 (83–109, MW 2988.38 g/mol) with a mutation (D100 → L) that increases its affinity for HLA-II type molecules associated to RA. A bioanalytical method, based on LC–MS/MS analysis, in the SRM mode was developed and fully validated to quantify this peptide in human plasma. An internal standard with the same amino acid sequence but labeled with three (13C615N2)-Lys residues was used for quantitation. The method provides a linear range from 1.5 to 48 ng/mL (without matrix effect and carry over) and an accuracy and precision good enough for monitoring more than 80% of the AUC of the PK profile in a phase I clinical trial. The peptide was administered subcutaneously in three dose levels (1, 2.5 and 5 mg) not normalized to the body weight of patients with RA. The low doses imposed an analytical challenge; however, a LLOQ of 1.5 ng/mL enabled the PK analysis. The Cmax, reached at 0.5 h, showed a great variability, that was most likely due to the non-normalized doses; the proposed mechanism for this peptide; and the variability between patients. A rapid clearance of this peptide (4–6 h) is advantageous for an immunomodulatory drug, because the therapeutic schedule requires repeated dosages to restore peripheral tolerance.