Purpose: The identification of specific target molecules and their appropriate cut-off values is crucial for the successful development of in vitro diagnostics. Salivary biomarkers, such as interleukin-1β (IL-1β) and matrix metalloproteinase-8 (MMP-8), have been investigated for their potential in diagnosing periodontal disease. This study aimed to evaluate the efficacy of IL-1β and MMP-8 as diagnostic tools and to establish their cut-off values for distinguishing active types of periodontal disease from healthy conditions. Methods: We conducted a prospective cohort study involving 47 participants, who were divided into 2 groups: the control group consisted of 24 individuals with healthy or mild periodontal conditions (stages I–II of periodontal disease), and the periodontal group comprised 23 individuals with moderate to severe periodontal disease (stages III–IV). Saliva samples were collected from all participants, followed by an oral examination, periodontal charting, and radiographic assessments. The salivary concentrations of IL-1β and MMP-8 were quantified using enzyme-linked immunosorbent assays. Results: Significant differences in salivary levels of IL-1β and MMP-8 were observed between the control group and those with periodontal disease. However, after adjusting for age and sex, these differences were not statistically significant for IL-1β. In contrast, the differences remained significant for MMP-8 in participants classified as having an active type of periodontal disease, specifically those in the periodontal group with more than 10% bleeding on probing. By setting a cut-off value of 200 ng/mL for salivary MMP-8, the sensitivity and specificity for diagnosing active periodontal disease were determined to be 70% and 86.5%, respectively. Conclusions Our findings indicate that salivary MMP-8, using a cut-off value of 200 ng/ mL, serves as a reliable biomarker for identifying active periodontal disease. This biomarker holds potential for further development into a diagnostic tool in in vitro settings, which could improve the early detection and management of periodontal disease.