Aim Zebrafish larvae with their simplified pronephros are an ideal model to study glomerular physiology. Although several groups use zebrafish larvae to assess glomerular barrier function, temporary or slight changes are still difficult to measure. The aim of this study was to investigate the potential of in vivo two‐photon microscopy (2‐PM) for long‐term imaging of glomerular barrier function in zebrafish larvae. Methods As a proof of principle, we adapted the nitroreductase/metronidazole model of targeted podocyte ablation for 2‐PM. Combination with a strain, which expresses eGFP‐vitamin D‐binding protein in the blood plasma, led to a strain that allowed induction of podocyte injury with parallel assessment of glomerular barrier function. We used four‐dimensional (4D) 2‐PM to assess eGFP fluorescence over 26 h in the vasculature and in tubules of multiple zebrafish larvae (5 days post‐fertilization) simultaneously. Results By 4D 2‐PM, we observed that, under physiological conditions, eGFP fluorescence was retained in the vasculature and rarely detected in proximal tubule cells. Application of metronidazole induced podocyte injury and cell death as shown by TUNEL staining. Induction of podocyte injury resulted in a dramatic decrease of eGFP fluorescence in the vasculature over time (about 50% and 90% after 2 and 12 h respectively). Loss of vascular eGFP fluorescence was paralleled by an endocytosis‐mediated accumulation of eGFP fluorescence in proximal tubule cells, indicating proteinuria. Conclusion We established a microscopy‐based method to monitor the dynamics of glomerular barrier function during induction of podocyte injury in multiple zebrafish larvae simultaneously over 26 h.