Whole plant chlorophyll fluorescence imaging is a powerful tool for non-destructive analysis of photosynthesis. Analysis of such images requires software that is able to process and calculate photosynthetic parameters per plant pixel. PlantCV is an open-source, Python-based library of image analysis tools for plant science. Previous versions of PlantCV included tools to analyze photosynthetic efficiency data, but recent developments to the photosynthesis subpackage have expanded to include more photosynthetic parameters based on chlorophyll fluorescence and spectral indices. This paper highlights the newest updates to the photosynthesis package of PlantCV and discusses applications of these tools on a sorghum dataset that was imaged with a PhenoVation CropReporter system.