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Abstract Amazônia is a species-rich region of immense importance to Earth’s water and carbon cycling. Photosynthesis drives the global carbon cycle, so understanding photosynthetic differences across diverse landscapes is a key task of ecophysiology and ecosystem science. Unfortunately, due to physiological and logistical constraints, ground-based photosynthesis data in Amazônia remain scarce and the ‘traditional’ steady-state (SS) method of gas exchange is slow and inefficient. The Dynamic Assimilation™ Technique (DAT) promises a new way to perform A/Ci curves rapidly without requiring SS conditions. Thus far, this technique has only been validated in greenhouse or agricultural-field-grown species and has yet to be tested in forest trees of diverse physiology morphology and environmental adaptation. To test the utility of the DAT in a complex tropical forest ecosystem, we compared the DAT with the SS method in 13 Amazonian trees in situ. We found strong agreement between Vcmax from DAT curves and SS curves, while Jmax was underestimated in DAT curves. We conclude that the DAT provides a robust and rapid estimation of Vcmax. We also identified diverse and unexpected DAT curve shapes among some trees, including the presence of an ‘overshoot’ in assimilation beyond model-derived ribulose-1,5-bisphosphate (RuBP) regeneration limitations. The presence of overshoot may elucidate microclimate and species differences in RuBP regeneration rates and emphasizes the considerable importance of DAT curve protocol specifications, such as the effect of ramp rate and direction on Jmax and TPU. Overall, the DAT saved time relative to the SS method and proved to be an effective and rapid method for quantifying Vcmax in tropical trees.