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Background: Doxofylline (DXE) is a novel methylxanthine derivative used in the treatment of asthma and Chronic Obstructive Pulmonary Diseases (COPD). Therapeutic Drug Monitoring (TDM) has been proposed in adults, while the adapted analytical method and TDM data are still missing in children. Methods: A highly sensitive and stability indicating High-Performance Liquid Chromatography (HPLC) method of DXE with caffeine as the internal standard, was developed and validated by separating its metabolites, β-Hydroxyethyltheophylline (HPE) and Theophylline (TPE). HPLC separation is achieved on C18 column connected to an ultraviolet detector (276 nm), using acetonitrile and ultra-pure water in a gradient mode of elution at a flow rate of 0.9 mL/min at 25°C. A liquid-liquid extraction method using ethyl acetate was developed with a small sample volume of plasma of 50 μL. Trough concentration was monitored in children receiving DXE therapy. Results: The method was linear over the concentration ranges from 0.4-20 μg/mL for DXE, HPE and TPE, respectively, in plasma. The limits of quantification were 0.4 μg/mL. Intra- and interday coefficients of variation did not exceed 6.5%, and the accuracy ranged from 94.9% to 112.5%. A total of 39 children (mean age of 1.8 years, range: 0.3-5.7 years) were included. The pediatric patients had detectable DXE concentrations with a mean value of 1.78 μg/mL (range from 0.49 to 6.36 μg/mL), and HPE measurable concentrations with a mean value of 0.52 μg/mL (range from 0.40 to 0.82 μg/mL), while the TPE could not be measured in any patient. Conclusion: A sensitive, reliable, and adapted HPLC method has been developed for the simultaneous analysis of DXE and its metabolites in children. The DXE and its metabolites trough concentrations showed large inter-individual variability.