In this work, we developed a measurement system that uses LEDs to estimate multiple components such as urea and creatinine in spot urine samples using near-infrared spectroscopy, considering future transition to LED light sources. In this study, we chose LEDs with 10 standard wavelengths (1400–2300 nm, in 100 nm increments). A multiple regression analysis using all combinations of 10 wavelengths was performed. We prepared glucose-added urine samples (GAU, urine samples from 10 healthy adults, each mixed with glucose). Wavelength selection was performed by comparing the minimum standard error of prediction (SEP, calculated from actual concentration and predicted concentration) for each wavelength combination. We obtained high accuracy for estimating urinary urea and creatinine levels (SEP: 42.4 mg/dl and 7.34 mg/dl, respectively) using four wavelengths for urea including two wavelengths showing negative absorbance, and five wavelengths for creatinine. Furthermore, an extremely high correlation coefficient (γ > 0.99) was obtained for both components. We calculated urea concentration, creatinine concentration, and urea-to-creatinine ratio using this optical, reagentless method. The low SEP and high γ show that our method is suitable for practical determination of urea-to-creatinine ratio. Thus, this method of analyzing urine samples using NIR spectroscopy can be used to assess protein intake in CKD patients.