Fish photolocomotor behavioral response (PBR) studies have become increasingly prevalent in pharmacological and toxicological research to assess the environmental impact of various chemicals. There is a need for a standard, reliable statistical method to analyze PBR data. The most common method currently used, univariate analysis of variance (ANOVA), does not account for temporal dependence in observations and leads to incomplete or unreliable conclusions. Repeated measures ANOVA, another commonly used method, has drawbacks in its interpretability for PBR study data. Because each observation is collected continuously over time, we instead consider each observation to be a function and apply functional ANOVA (FANOVA) to PBR data. Using the functional approach not only accounts for temporal dependency but also retains the full structure of the data and allows for straightforward interpretation in any subregion of the domain. Unlike the traditional univariate and repeated measures ANOVA, the FANOVA that we propose is nonparametric, requiring minimal assumptions. We demonstrate the disadvantages of univariate and repeated measures ANOVA using simulated data and show how they are overcome by applying FANOVA. We then apply one-way FANOVA to zebrafish data from a PBR study and discuss how those results can be reproduced for future PBR studies.