Optophysiological imaging methods can be used to record the activity in vivo of groups of neurons from particular areas of the nervous system (e.g. the brain) or of cell cultures. Such methods are used, for example, in the spatio-temporal coding and processing of sensory information. However, the data generated by optophysiological methods must be processed carefully if relevant results are to be obtained. The raw fluorescence data must be digitally filtered and analyzed appropriately to obtain activity maps and fluorescence time course for single spots.We used a Matlab® environment to implement the necessary procedures in a user-friendly manner.We developed OpenFluo, a program for people inexperienced in optophysiological methods and for advanced users wishing to perform simple, rapid data analyses without the need for complex, time-consuming programming procedures. This program will be made available as stand-alone software and as an open-source Matlab® tool. It will therefore be possible for experienced users to integrate their own routines. We validated this software by assessing its ability to process both artificial recordings and real biological data corresponding to recordings of the honeybee brain.