Abstract: An improved and fast reversed phase high performance liquid chromatography(HPLC) method with high efficiency chromatography column, pecosphere C₁₈, 3 μm, 3.3 cm×4.6 mm, I.D(Perkin Elmer, USA) for determination of theophylline in human serum is described. The method uses a 0.1 mol/L sodium acetate buffer(pH=4.5) methanol mobile phase(70:30, V/V) with a flow rate of 0.5 mL/min and uses paracetamol as internal standard(IS). Theophylline and IS are extracted from serum using a mixture of trichloromethane: isopropanol(95:5, V/V). Quantitation is by UV absorbance at 270 nm. The intra day and inter day determination of precision for theophylline(5.27 μg/mL) varies from 2.5% to 5.4% and 2.1% to 5.0% for theophylline(10.50 μg/mL), respectively. The average recovery is more than 87.3%(n=5). Analysis time is approximately 3 min for theophylline and internal standard, it is faster than the time of reported methods(analysis times are approximately 7.3～28.4 min).