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Simple Summary: The gas exchange system presented herein integrates open-flow/force ventilation, LED technology, and micropropagation to determine the impact of environmental factors (e.g., [CO2], sucrose, light intensity) on the photosynthetic capacity of cultured plantlets. This system was developed and tested on Cannabis sativa L., an emerging crop of high economic value, for which micropropagation has become an important aspect of production. Since conventional micropropagation avenues can minimize photosynthetic performance, this system offers fresh opportunities to examine the role of light signaling and photosynthesis in micropropagation to investigate and overcome in-vitro-associated morphophysiological disorders. By maintaining [CO2] at controlled levels (400 and 1200 ppm) with calibrated light intensities, photosynthetic light response curves were prepared based on net carbon exchange rates (NCERs) to paint a picture of the dynamic, combinational influences of irradiance, [CO2], and additional factors on photosynthetic performance. Additionally, NCERs were continuously monitored during a 24 h light/dark period under standard conditions to provide estimates of relative growth rates (daily C-gain). Thus, a system is presented with the ability to answer questions about the nature of in vitro plant physiology related to carbon dynamics, that would otherwise be difficult to assess. Supplemental sugar additives for plant tissue culture cause mixotrophic growth, complicating carbohydrate metabolism and photosynthetic relationships. A unique platform to test and model the photosynthetic proficiency and biomass accumulation of micropropagated plantlets was introduced and applied to Cannabis sativa L. (cannabis), an emerging crop with high economic interest. Conventional in vitro systems can hinder the photoautotrophic ability of plantlets due to low light intensity, low vapor pressure deficit, and limited CO2 availability. Though exogenous sucrose is routinely added to improve in vitro growth despite reduced photosynthetic capacity, reliance on sugar as a carbon source can also trigger negative responses that are species-dependent. By increasing photosynthetic activity in vitro, these negative consequences can likely be mitigated, facilitating the production of superior specimens with enhanced survivability. The presented methods use an open-flow/force-ventilated gas exchange system and infrared gas analysis to measure the impact of [CO2], light, and additional factors on in vitro photosynthesis. This system can be used to answer previously overlooked questions regarding the nature of in vitro plant physiology to enhance plant tissue culture and the overall understanding of in vitro processes, facilitating new research methods and idealized protocols for commercial tissue culture. [ABSTRACT FROM AUTHOR]