In this study, we developed and validated two reliable high-performance liquid chromatography (HPLC) methods for the qualitative detection of six oral β-lactams, which are commonly used in pediatric patients with acute respiratory infections (ARIs). Two distinct reverse-phase chromatographic separations of six β-lactams were obtained. Four β-lactams (cefadroxil, cephalexin, cefaclor, cefixime) in urine were separated using a gradient program with a mobile phase consisting of K2HPO4 buffer (20 mM, pH 2.8) and acetonitrile on a LichroCART 250x4.6 mm, Purospher STAR C18 end-capped (5μm) column. Two remained β-lactams (amoxicillin, cefuroxime) were analyzed using a gradient elution with the mobile phase containing K2HPO4 buffer (20 mM, pH 3.0) and acetonitrile on a LichroCart® Purospher Star C8 end-capped column (5 μm, 125x4.6 mm) column. Good linearity within the range of 0.3-30 μg/mL for cefadroxil, cephalexin, cefaclor and cefixime, and 0.2-20 μg/mL for amoxicillin and cefuroxime, was attained. The precisions were less than 14%. The accuracies ranged from 85.87-102.8%. The two validated methods were then applied to determine these six antibiotics in 553 urine samples of pediatric patients with ARIs. As resulted, 32.2% were positive with one or more of six tested β-lactams. Cefixime was the most commonly detected agent, accounting 9.8% of enrolled patients.