Nephrotic syndrome is characterized by urinary excretion of plasma proteases or proteasuria. There is a lack of data on the quantity, activity status and identity of these aberrantly filtered proteases. We established a fluorescence-based substrate assay to quantify protease activity in urine samples from healthy and nephrotic humans and mice. Protease class activity was determined after addition of specific inhibitors. Individual proteases were identified by tandem mass spectrometry (MS/MS). In spot urine samples from 10 patients with acute nephrotic syndrome of various etiology, urinary protease activity was significantly increased compared to that of healthy persons (753 ± 178 vs. 244 ± 65 relative units, p < 0.05). The corresponding proteases were highly sensitive to inhibition by the serine protease inhibitors AEBSF (reduction by 85 ± 6% and 72 ± 8%, respectively) and aprotinin (83 ± 9% vs. 25 ± 6%, p < 0.05). MS/MS of all urinary proteins or after AEBSF purification showed that most of them were active serine proteases from the coagulation and complement cascade. These findings were recapitulated in mice, pointing to a similar pathophysiology. In conclusion, nephrotic syndrome leads to increased urinary excretion of active plasma proteases which can be termed proteasuria. Serine proteases account for the vast majority of urinary protease activity in health and nephrotic syndrome. Significance statement: In this study, we found that nephrotic urine samples of humans and mice have a significantly increased protease activity compared to healthy urine samples, using a universal pentapeptide substrate library. This was driven by increased excretion of aprotinin-sensitive serine proteases. With tandem mass spectrometry, we provide a comprehensive and systematic overview of all urinary proteases or the “urine proteasome”. We identified renally expressed proteases in health and addition of proteases from the coagulation and complement cascade in the nephrotic state. These results set the basis to study the role of urinary proteases at both health and nephrotic syndrome to find diagnostic markers of renal disease as well as possible therapeutic targets.