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The problem of entrainment is central to circadian biology. In this regard, Drosophila has been an important model system. Owing to the simplicity of its nervous system and the availability of powerful genetic tools, the system has shed significant light on the molecular and neural underpinnings of entrainment. However, much remains to be learned regarding the molecular and physiological mechanisms underlying this important phenomenon. Under cyclic light/dark conditions, Drosophila melanogaster displays crepuscular patterns of locomotor activity with one peak anticipating dawn and the other anticipating dusk. These peaks are characterized through an estimation of their phase relative to the environmental light cycle and the extent of their anticipation of light transitions. In Drosophila chronobiology, estimations of phases are often subjective, and anticipation indices vary significantly between studies. Though there is increasing interest in building flexible analysis tools in the field, none incorporates objective measures of Drosophila activity peaks in combination with the analysis of fly activity/sleep in the same program. To this end, we have developed PHASE, a MATLAB-based program that is simple and easy to use and (i) supports the visualization and analysis of activity and sleep under entrainment, (ii) allows analysis of both activity and sleep parameters withinmore »user-defined windows within a diurnal cycle, (iii) uses a smoothing filter for the objective identification of peaks of activity (and therefore can be used to quantitatively characterize them), and (iv) offers a series of analyses for the assessment of behavioral anticipation of environmental transitions.