Plants encode for >100 metalloproteases representing >19 different protein families. Tools to study this large and diverse class of proteases have not yet been introduced into plant science. Here, we used hydroxamate-based photoaffinity probes to explore plant proteomes for metalloproteases. We detected labeling of 23 metalloproteases in leaf extracts of the model plant Arabidopsis thaliana that belong to eight unrelated metalloprotease families and localize to different subcellular compartments. We identified several chloroplast localized FtsH proteases; vacuolar aspartyl aminopeptidase DAP1; peroxisomal metalloprotease PMX16; and many cytosolic metalloproteases. We also identified non-proteolytic metallohydrolases involved the release of auxin and in the urea cycle. Studies on tobacco plant Nicotiana benthamiana infected with the bacterial plant pathogen Pseudomonas syringae uncovered PRp27, a secreted protein with anticipated metalloprotease activity. PRp27 depletion increases susceptibility, whilst its overexpression increases resistance, dependent on the presence of active site residues. Collectively, these studies uncover the strength of hydroxamate-based probes to study metalloenzymes in plants