Owing to its high water use efficiency (WUE), cactus pear (Opuntia ficus-indica) is an alternative for producing biomass with bioenergetic purpose in arid and semi-arid zones. In Chile, irrigated cactus pear plantations with such purpose are being planned and germplasm with high WUE, but also high radiation use efficiency (RUE) needs to be screened. Because of the morphology and metabolism of cactus pear cladodes, the use of infrared gas analyzers (IRGA) for studying the light response of CO2 assimilation is very difficult. Therefore, most studies are carried out by measuring variations in cladode daily titratable acidity (DTA). An alternative is to estimate CO2 assimilation from the light response of linear electron transport rate (ETR), which can be estimated from chlorophyll fluorescence measurements. We estimated net CO2 assimilation of single cactus pear cladodes emerging from rooted cladode fragments grown in sun and in 50% shade in a greenhouse. Estimations were based on DTA, IRGA measurements and a model based on the light response curve of ETR and a cladode respiration sub-model depending on temperature and growth rate. DTA and IRGA measurements were in excellent agreement but the model underestimated net CO2 assimilation, which may be attributed to a poor estimation of respiration, and overestimated cladode growth rate, probably because root growth was not considered for assessing growth. Nonetheless, modeled net CO2 assimilation and growth rate of cladodes exhibited high coefficients of determination, when compared to measured rates. This indicates that chlorophyll fluorescence measurement can be considered as a good tool for assessing RUE of cactus pear germplasm provided a further development of the model.