Despite multiple efforts to target an improvement in clinical outcomes of patients with end-stage renal disease, several challenges must still be addressed. Dialysis patients are at a high risk for complications, as reflected by increasing mortality rates. The objective of this study is to assess the impact of the application of dialyzers with varying permeability profiles on the removal of cell-activating substances from the blood of hemodialysis (HD) patients. Dialysate samples were collected using Revaclear 400 (RC) and MCO-Ci400 (MCO-CI). Total protein and solute marker concentrations were determined for the concentrated sample. The response of tubular epithelial cells (TECs) to the dialysate samples was assessed via measurement of interleukin 6, cell viability, and morphology. Proteomic analysis of the dialysate samples was performed using liquid chromatography coupled to tandem mass spectrometry. Treatment of TECs with the MCO-CI dialysate resulted in significantly decreased cell viability compared with the RC dialysate. TECs incubated with samples from MCO-CI lost their typical brick-like shape and cell-cell connections. Proteomic analysis of dialysate samples indicated multiple pro-apoptotic and pro-inflammatory proteins, supporting the observed phenotype. Additionally, application of the MCO-CI dialyzer allowed for more efficient removal of proteins associated with advanced chronic kidney disease stages. Collectively, the use of dialyzer with a higher permeability profile enabled more efficient removal of cell-activating and toxic substances from the blood of HD patients. However, a further large-scale study is needed to address benefits and associated risks for patients.