Senescence is the highly regulated last developmental phase of plant organs and tissues, and is optimized to allow nutrient remobilization to surviving plant parts, such as seeds of annual crops. High leaf carbohydrate to nitrogen (C : N) ratios have been implicated in the induction or acceleration of the senescence process. *A combination of phloem interruption in mature leaves (by steam-girdling, leading to carbohydrate accumulation from photosynthesis) and varied nitrate supply was used to analyse correlations between metabolite levels, leaf senescence parameters and induction of protease genes and proteolytic activities. *Its strong induction under conditions characterized by high C : N ratios, negative correlation of its transcript levels with chlorophylls and nitrates, its strong induction during developmental leaf senescence and its predicted localization to a lytic vacuolar compartment indicate that, among the genes tested, a family C1A cysteine protease is most likely to participate in bulk protein degradation during barley leaf senescence. *While all the genes analysed were selected based on upregulation during leaf senescence in a previous transcriptomic study, a considerably more detailed picture of protease gene regulation emerged from the data presented here, underlining the usefulness of this experimental approach for further (functional) protease characterization.