Background Gastrointestinal motility measurements in mice are currently performed under suboptimal conditions, as these nocturnal animals are measured during light conditions. In addition, other stressors, like individual housing, placement in a new cage during observation, and lack of bedding and cage enrichment cause animal discomfort and might contribute to higher variability. Here we aimed to develop a refined method of the widely‐used whole‐gut transit assay. Methods Wildtype mice (N = 24) were subjected to the standard or refined whole‐gut transit assay, either with or without a standardized slowing in gastrointestinal motility induced by loperamide. The standard assay consisted of a gavage with carmine red, observation during the light period and individual housing in a new cage without cage enrichment. For the refined whole‐gut transit assay, mice were gavaged with UV‐fluorescent DETEX®, observed during the dark period, while pairwise housed in their home cage with cage enrichment. Time until excretion of the first colored fecal pellet was assessed, and pellets were collected to assess number, weight, and water content. Key Results The DETEX®‐containing pellets were UV‐detectable, allowing to measure the mice in their active period in the dark. The refined method caused less variation (20.8% and 16.0%) compared to the standard method (29.0% and 21.7%). Fecal pellet number, weight, and water content was significantly different between the standard and refined method. Conclusions & Inferences This refined whole‐gut transit assay provides a reliable approach to measure whole‐gut transit time in mice in a more physiological context, with reduced variability compared to the standard method.