In vivo animal experiments are critical in the process of finding and developing new treatments for children with CNS tumors. Cerebral microdialysis, which enables researchers to measure drug concentrations in the brain or tumor tissue of unanesthetized mice, is a highly specialized procedure that provides valuable information that cannot be gained by using an in vitro system. When designing any in vivo animal study, 3 Rs principles (replacement, reduction, and refinement) must be considered to ensure that the highest standards of care are followed. As part of the refinement process, the objectives of this study were to collect behavioral monitoring data from mice undergoing cerebral microdialysis, to identify any behaviors predictive of significant pain or distress that could affect the animal's welfare, and to use these data to refine the existing monitoring checklist and schedule for its use by others performing this procedure. We developed a monitoring checklist for assessing wellbeing and distress of mice during cerebral microdialysis experiments. Comparison of 79 mice that underwent cerebral microdialysis experiments with a control group of 20 mice revealed that cerebral microdialysis and tethering of mice are well tolerated for as long as 24 h with only minor evidence of stress.