Upon a drastic change in environmental illumination, zebrafish larvae display a rapid locomotor response. This response can be simultaneously tracked from larvae arranged in multi-well plates. The resulting data have provided new insights into neuro-behaviour. The features of these data, however, present a challenge to traditional statistical tests. For example, many larvae display little or no movement. Thus, the larval responses have many zero values and are imbalanced. These responses are also measured repeatedly from the same well, which results in correlated observations. These analytical issues were addressed in this study by the generalized linear mixed model (GLMM). This approach deals with binary responses and characterizes the correlation of observations in the same group. It was used to analyze a previously reported dataset. Before applying the GLMM, the activity values were transformed to binary responses (movement vs. no movement) to reduce data imbalance. Moreover, the GLMM estimated the variations among the effects of different well locations, which would eliminate the location effects when two biological groups or conditions were compared. By addressing the data-imbalance and location-correlation issues, the GLMM effectively quantified true biological effects on zebrafish locomotor response.