Current tropospheric ozone (O3) background concentrations have significant adverse effects on wheat. O3 generally induces oxidative damages and premature leaf senescence leading to important yield losses. As vacuolar Endoproteases (EP) are involved in both maintaining cell longevity during abiotic stresses and performing efficient nitrogen remobilization during leaf senescence, we aimed to characterize acidic endoproteolytic activities involved during natural and O3-induced flag leaf senescence in wheat. Two winter wheat cultivars (cvs.) with different O3 sensitivity, Soissons and Premio, were grown in the field at the experimental farm of AgroParisTech (Thiverval Grignon, France) and exposed to ambient (AOT40 = 1.58 ppm.h) and semi-controlled chronic O3 concentrations (AOT40 = 3.71 ppm.h), from pre-anthesis to grain harvest. Flag leaf total EP activity and the major catalytic classes involved were determined using azocasein and class-specific inhibitors, respectively. Activity-based protein profiling (ABPP) was used to identify the major active cysteine proteases involved in natural and O3-induced leaf senescence. Our analysis revealed that natural leaf senescence and O3 stress induced a stimulation of endoproteolytic activities. In both cases, global leaf endoproteolytic activity was mostly due to papain-like cysteine proteases (PLCPs). ABPP analyses using DCG-04 revealed an increase in active PLCP contents in wheat flag leaf in response to O3 for both cvs. A pull-down of active PLCPs in the naturally senescing and O3-exposed flag leaf of cv. Premio was carried out and active PLCPs were identified using LC-MS/MS. Mass spectrometry analyses revealed that triticain was the major active PLCP in senescing flag leaf and also the only PLCP whom abundance was increased by O3 stress. Gene expression analysis by qPCR indicated that triticain activity could be regulated at the transcriptional level.