Patients with nephrotic syndrome (NS) suffer from urinary loss of albumin. As a cause, previous studies focused on the glomerular filter rather than analyzing the molecular properties of albumin itself. Later one was initiated by clinical observations indicating unexplained molecular alterations of human serum albumin (HSA) in an NS pediatric patient. Therefore, we examined serum from eight pediatric patients with steroid-sensitive and -resistant NS and compared it with serum from healthy subjects as well as commercial HSA. We used dynamic and electrophoretic light scattering to characterize the protein size and effective surface charge and electron paramagnetic resonance spectroscopy to measure the local environment and binding dynamics of up to seven fatty acids associated with HSA. Our findings suggest that pronounced differences in binding behavior and surface charge of HSA could enhance their filtration through the GBM, leading to direct toxicity of HSA to podocytes.