Introduction The concentration of substances, including urea, creatinine, and uric acid, can be used as an index to measure toxic uremic solutes in the blood during dialysis and interdialytic intervals. The on-line monitoring of toxin concentration allows for the clearance measurement of some low-molecular-weight solutes at any time during hemodialysis.The aim of this study was to determine the optimal wavelength for estimating the changes in urea, creatinine, and uric acid in dialysate, using ultraviolet (UV) spectroscopy. Materials and Methods In this study, nine uremic patients were investigated, using on-line spectrophotometry. The on-line absorption measurements (UV radiation) were performed with a spectrophotometer module, connected to the fluid outlet of the dialysis machine. Dialysate samples were obtained and analyzed, using standard biochemical methods. Optimal wavelengths for both creatinine and uric acid were selected by using a combination of genetic algorithms (GAs), i.e., GA-partial least squares (GA-PLS) and interval partial least squares (iPLS). Results The Artifitial Neural Network (ANN) sensitivity analysis determined the wavelengths of the UV band most suitable for estimating the concentration of creatinine and uric acid. The two optimal wavelengths were 242 and 252 nm for creatinine and 295 and 298 nm for uric acid. Conclusion It can be concluded that the reduction ratio of creatinine and uric acid (dialysis efficiency) could be continuously monitored during hemodialysis by UV spectroscopy.Compared to the conventional method, which is particularly sensitive to the sampling technique and involves post-dialysis blood sampling, iterative measurements throughout the dialysis session can yield more reliable data.