A fundamental challenge in plant physiology is independently determining the rates of gross O2 production by photosynthesis and O2 consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O2 production (NOP and GOP, respectively) by labeling ambient O2 with 18O while leaf water was unlabeled. Here, we introduce a new method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas exchange measurement. The petiole is immersed in water enriched at a d18O of ~9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers 18O from H2O to O2. GOP is calculated from the increase in d18O of O2 as air passes through the cuvette. NOP is determined from the increase in O2/N2. Both terms are measured by isotope ratio mass spectrometry. CO2 assimilation and other standard gas exchange parameters are also measured. Reproducible measurements are made on a single leaf for more than 15 hours. We used this method to measure the light response curve of NOP and GOP in Phaseolus vulgaris at 21% and 2% O2. We then used these data to calculate the O2/CO2 ratio of net photosynthesis, the light response curve of mesophyll conductance, and the inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique presented as a unique tool to study and understand leaf physiological traits.