Aims: The current study aimed that first morning voided (FMV) urinary gonadotropin measurements could be used as a noninvasive alternative to the gonadotropin-releasing hormone (GnRH) test in the assessment of the hypothalamic-pituitary-gonadal function in children. Methods: In a multi-center study, we compared FMV urinary gonadotropin concentrations with GnRH-stimulated serum gonadotropin levels in 140 girls aged 7-9 years who were evaluated for pubertal development, height acceleration, and bone maturation. Urinary and serum LH and FSH were determined by time-resolved sandwich fluoroimmunoassays (Delfia hLH Spec and Delfia hFSH; Wallac Oy, Turku, Finland). The relationship between FMV urinary gonadotropin concentrations and GnRH test results was assessed. Results: FMV urinary LH (U-LH) and FMV urinary LH to FSH ratio (U-LH to U-FSH ratio) were significantly positively correlated with peak LH in GnRH stimulation test (r=0.49, P=0.030 and r=0.070, P0.001). In receiver operating characteristic (ROC) curve analyses, the level of 0.89 for FMV U-LH was determined to be a possible cutoff to predicting a pubertal GnRH stimulation test result, with a sensitivity of 90.9%, a specificity of 77.8%, and area under the curve 0.49-1.00 ( P=0.006). Conclusion: FMV U-LH determination may be an alternative assessment for pubertal development and its disorders, reducing the need for invasive GnRH stimulation tests.