Background: Gut dysbiosis has been implicated in the development and progression of chronic kidney disease (CKD) by the generation of uremic toxins, such as the protein-bound solutes p-cresyl sulfate (pCS) and indoxyl sulfate (IXS). In this study, we evaluate the associations of total (t) and free fractions (f) of pCS and IXS with the Kidney Disease: Improving Global Outcomes (KDIGO) glomerular filtration rate (GFR; G) categories using contemporary GFR estimation equations. Methods: Liquid chromatography/tandem mass spectrometry has been applied for the identification and quantification of pCS and IXS. Results: We evaluated 80 patients (55 ± 13 years); 41 male. We found a statistically significant increase of pCSt, pCSf, IXSt and IXSf across the CKD spectrum as defined by GFR categories (P < 0.001). A significant negative correlation was observed between estimated glomerular filtration rate (eGFR, any equations) and plasma concentrations of pCS and IXS (all P values < 0.001). For example we reported correlation between eGFR 2021 Chronic Kidney Disease Epidemiology Collaboration, CKD-EPI and pCSt (Spearman’s coefficient Rho [r] = -0.690), pCSf (r = -0.744), IXSt (r = -0.754) and IXSf (r = -0.819). Conclusion: We found that the free fraction as well as total concentrations of pCS and IXS were negatively correlated with estimated GFR (all equations) in CKD patients. The significant increase of pCS and of IXS across the CKD spectrum suggests that both protein-bound solutes may be used as surrogate markers of renal function.