Although the rates of gross protein depolymerization are central in litter decomposition, they cannot be measured directly by any existing method. Here we report the development of a novel assay to quantify gross protein depolymerization (i.e. gross amino acid production) based on a 15N isotope pool dilution technique. The assay is based on the concurrent labeling of the pool of 18 proteinogenic amino acids, which are present in a free form in litter, and the measurement of 15N:14N ratios in the individual amino acids by GC–MS over time. The method proved to be highly linear, precise and sufficiently sensitive. We tested the applicability of the novel method on a litter decomposition experiment, in which we could demonstrate that gross protein depolymerization exceeded gross N mineralization by >8 fold indicating that only a small fraction of amino acids released by extracellular enzymes was actually mineralized to ammonium. Moreover, the results provide evidence that protein depolymerization was limited by protein availability or accessibility, not by the size of protease pool.