Podocyte injury is a major cause of chronic kidney disease. However, understanding podocyte‐specific responses at a systems level is difficult when glomerular cellular composition is altered by loss of podocytes or increase of extracellular matrix. We developed and applied proteome analysis to very small sample amounts to podocyte fractions isolated from single mice after induction of glomerular disease and to single glomeruli from patients with Focal segmental glomerulosclerosis (FSGS). Transcriptome and proteome analysis of glomeruli from patients with FSGS revealed an underrepresentation of podocyte‐specific genes and proteins in late‐stage disease. To circumvent this problem, we utilized two commonly used mouse models of chemically‐induced FSGS and performed proteomic analysis of pure podocyte fractions in early disease states. We observed perturbations of propagating pathways of glomerular disease such as Jak‐Stat signaling, central carbon metabolism such as TCA cycle enzymes, and mechanical stress response. Multi‐layered priorization approaches identified a co‐regulation of proteinuria with the mechanosensor protein Filamin‐B. Podocytes in human biopsies, single glomeruli from proteinuric rats and podocytes undergoing mechanical stress, showed increased expression of Filamin‐B, suggesting a role in detrimental stress responses. A nephrocyte‐specific depletion of Cher, the Filamin homolog in Drosophila, showed an altered filtration capacity while nephrocyte morphology was unaffected. This study identified conserved mechanisms of the podocyte stress response by analyzing ultrasensitive proteomes from human glomerular FSGS tissue and purified native mouse podocytes during early disease stages. The pipeline put forward here enables systematic comparisons of large‐scale proteomics data and phenotype to protein correlation. Support or Funding Information: MMR was supported by the DFG (Ri2811/1 and Ri2811/2).