The voiding spot assay (VSA) on filter paper is an increasingly popular method for studying lower urinary tract physiology in mice. However, the ways VSA are performed differ significantly between laboratories, and many variables are introduced compared to the mouse's normal housing situation. Rodents are intelligent social animals, and it is increasingly understood that social and environmental stresses have significant effects on their physiology. Surprisingly, little is known about whether change of environment during VSA affects mouse voiding, and what the best methodologies are for retaining 'natural' micturition patterns. It is well known that stress-related neuropeptide corticotropin-releasing factor (CRF) is significantly elevated and induces dramatic voiding changes when rodents encounter stresses. Therefore, we hypothesized that changes in the environmental situation could potentially alter voiding during VSA. We have examined multiple factors to test whether they affect female mouse voiding patterns during VSA, including cage type, cage floor, water availability, water bottle location, single or group housing, and different handlers. Our results indicate that mice are surprisingly sensitive to changes of cage type and floor surface, water bottle location, and single/group housing, each of which induces significant changes in voiding patterns, indicative of a stress response. In contrast, changing handler or four hours of water deprivation did not affect voiding patterns. Our data indicate that VSA should be performed under conditions as close as possible to the mouse's normal housing. Optimizing VSA methodology will be useful in uncovering voiding alterations in both genetic and disease models of lower urinary dysfunctions.