Purpose: To develop and validate a new low-cost high performance liquid  chromatography (HPLC) method for simultaneous analysis of theophylline (TH), guaifenesin (GF) and diphenhydramine hydrochloride (DH) in elixir dosage form.Methods: Chromatographic conditions were an isocratic elution with C18-Kromasil® column (250 x 4.6 mm, 5 μm), methanol-water (1:1, v/v, pH 3,0) as mobile phase, flow rate 1.0 ml/min and UV detector at λ 218 nm. The method was validated for selectivity, linearity, LOD-LOQ, precision, and accuracy.Results: Retention time of TH, GF and DH was 3.3, 5.3 and 9.1 min, respectively. The method showed good selectivity, calibration curves were linear over the concentration range of 1.000 – 10.002 μg/mL, 0.801 – 8.008 μg/mL, and 0.251 – 2.514 μg/mL (r2 > 0.999). LOD was 0.1093, 0.16520, and 0.0706 μg/mL, while LOQ was 0.3645, 0.5506, and 0.2354 μg/mL for TH, GF and DH, respectively. Recovery accuracy was 99.77 - 101.10, 100.50 - 101.95 and 99.20 - 100.13 % for TH, GF and DH, respectively; precision (RSD) was < 2.0.Conclusion: The proposed method is highly selective, sensitive, precise, and  accurate, and would suitable for the simultaneous analysis of TH, GF, and DH in elixir dosage form. Since methanol is cheaper than acetonitrile, the application of the method may reduce the cost of analysis.Keywords: Simultaneous analysis, Theophylline, Guaifenesin, Diphenhydramine hydrochloride, Elixir