In leaf gas exchange measurements, cuticular conductance to water (gcw) is indistinguishable from and included in stomatal conductance to water vapor (gsw). Here we developed a simple technique to isolate gcw by directly measuring leaf intercellular CO2 concentration (Ci(m)) along with gas exchange during photosynthetic light induction. We derived stomatal conductance to CO2 (gsc(m)) from the Ci(m) independently of gsw. Plotting gsw against gsc(m) during the early induction phase within ~10 min, we found a highly linear relationship with a positive intercept. Assuming negligible cuticular CO2 transport, complete stomatal closure occurs when gsc(m)=0. Then, we considered the residual gsw (i.e., intercept) as gcw. Indeed, these gcw estimates succeeded in correcting the calculation. Our technique, owing to its robustness and increased throughput, will allow for more rapid screening of crops, more reliable gas exchange analysis, and more accurate prediction of plant function under natural environmental conditions.