A high-performance liquid chromatography–diode array detector method (HPLC-DAD) was developed and validated for the determination of tramadol in human saliva. Samples of saliva were prepared utilizing liquid–liquid extraction with hexane-ethyl acetate (4: 1, v/v). Propranolol was used as an internal standard (IS). The linear range and average recovery of tramadol were 0.25–4.00 µg/mL and 94.70%, respectively. The intra-and inter-assay precisions at low, intermediate and high concentrations were within 1.40–9.79% and 0.77–3.98%, respectively. Intra-and inter-assay accuracies were within 98.50–107.13% and 100.44–103.89%, respectively. The proposed method was successfully applied to determine tramadol in saliva concentrations in three healthy volunteers for 24 hours after administration of 100 mg oral doses of tramadol.