Background Normal gut function requires rhythmic and coordinated movements that are affected by developmental processes, physical and chemical stimuli, and many debilitating diseases. The imaging and characterization of gut motility, especially regarding periodic, propagative contractions driving material transport, are therefore critical goals. Previous image analysis approaches have successfully extracted properties related to the temporal frequency of motility modes, but robust measures of contraction magnitude, especially from in vivo image data, remain challenging to obtain. Methods We developed a new image analysis method based on image velocimetry and spectral analysis that reveals temporal characteristics such as frequency and wave propagation speed, while also providing quantitative measures of the amplitude of gut motion. Key Results We validate this approach using several challenges to larval zebrafish, imaged with differential interference contrast microscopy. Both acetylcholine exposure and feeding increase frequency and amplitude of motility. Larvae lacking enteric nervous system gut innervation show the same average motility frequency, but reduced and less variable amplitude compared to wild types. Conclusions & Inferences Our image analysis approach enables insights into gut dynamics in a wide variety of developmental and physiological contexts and can also be extended to analyze other types of cell movements. We present a new image analysis technique using image velocimetry and spectral analysis that returns quantitative measures of gut contraction strength, frequency, and wave speed that can be used to study gut motility and other cellular movements.