Enzymatic O sensors transduce the availability of O within the cell into a physiological, typically adaptive response. One such O -sensing enzymatic family is the N-terminal cysteine dioxygenases in plants (plant cysteine oxidases PCOs). In vitro kinetic studies have determined the O -sensing capacity of PCOs. Here we describe the rationale and experimental protocol for an assay with which the O sensitivity of Arabidopsis thaliana PCOs (AtPCOs) can be measured. We explain each step from the recombinant protein synthesis of AtPCOs to the steady-state kinetic assays of AtPCOs for primary substrate and O from which kinetic parameters can be derived. The same techniques can be applied to other N-terminal cysteine thiol dioxygenases, e.g. 2-aminoethanethiol dioxygenase (ADO), and similar principles can be applied to determine kinetic characteristics of other oxygenase enzymes towards O .