SummaryA reversed -phase HPLC method with UV detection for determination of chlorhexidine in gingival crevicular fluid (GCF) was optimized and validated, using chlorpheniramine as an internal standard. The chromatographic separation was performed on Discovery C18 HPLC column with 0.01 mol L -1 phosphate buffer (pH=3.0), triethylamine and acetonitrile (66:1:33, V/V/V), as mobile phase. Under the optimized HPLC conditions, linearity was obtained in the range of 0.5-5.0 µg mL -1 with LOD 0.07 µg mL -1 and LLOQ 0.5 µg mL -1 . The described method can be successfully applied for determination of chlorhexidine concentrations in GCF obtained from patients with chronic periodontal disease treated with PerioChip TM .