Background Urinary oxalate can provide important clues for the screening and monitoring of children with primary hyperoxaluria (PH), which is a potentially life-threatening condition. However, little effort has been devoted to improve the oxalate assay in recent years. We have proposed a reliable and cost-effective high-performance liquid chromatography (HPLC) method for urinary oxalate determination. Methods Urine specimens were centrifuged after one-step derivatization, and the supernatants were subjected to HPLC analysis. Results The method was validated with consistent linearity from 0.0625 to 2.0 mmol/L with coefficients of variation <= 7.73%, good recovery, low carryover, satisfactory sample stability, and analytical specificity. The lower limit of quantification and the limit of detection were 0.03130 and 0.0156 mmol/L, respectively. Imprecision values were <= 2.92% and <= 16.6% for externally and internally produced controls, respectively. The pediatric reference interval of spot urinary oxalate to creatinine ratios was established together with its application in screening of PH in patients with renal diseases, revealing its successful deployment in our laboratory. Conclusions This reliable HPLC method could serve as a significant tool to determine urinary oxalate levels for screening and monitoring of children with PH in routine clinical laboratories.