Kidney failure is a global health concern, exacerbated by the accumulation of uremic metabolites that persist despite conventional treatments such as hemodialysis (HD). A significant subset of these metabolites, termed protein-bound uremic toxins (PBUTs), bind tightly to human serum albumin (HSA), limiting their removal through traditional separation techniques. This dissertation identifies tryptophan, 4-ethylphenyl sulfate (4-EPS), and putrescine as PBUTs and investigates the binding mechanisms and thermodynamic interactions of them with HSA to address gaps in the literature and advance understanding of their role in disease progression. Using a combination of Isothermal Titration Calorimetry (ITC), Saturation Transfer Difference Nuclear Magnetic Resonance (STD-NMR), and Circular Dichroism (CD), the study identifies the binding sites, energetics, and driving forces behind the interactions of these metabolites with HSA. Tryptophan binds predominantly to Sudlow's site II, driven by hydrophobic interactions, with minimal impact on HSA conformation. 4-EPS exhibits dual-site binding at Sudlow's sites I and II, characterized by distinct thermodynamic profiles and competitive interactions influenced by structural similarities with other PBUTs. Conversely, putrescine demonstrates weak, non-specific interactions with HSA, challenging earlier assumptions. Furthermore, a longitudinal metabolomics study reveals that plasma levels of 74 metabolites in end-stage renal disease (ESRD) patients undergoing maintenance HD fluctuate significantly over 60 months. Among these, 43 metabolites increased in serum concentration, including 24 not previously associated with kidney failure, highlighting the limitations of current treatment strategies. This research contributes to the understanding of PBUT-HSA interactions, their implications for treatment inefficacy, and their role in systemic complications associated with kidney failure. By providing a foundation for future studies, this work advocates the development of advanced therapeutic strategies and dialysis technologies to mitigate the impact of uremic metabolites on patient outcomes.