Nest building and burrowing are highly motivated natural behaviors in rodents, and changes in these behaviors can serve as welfare assessment tools. In this study, we investigated: 1) the limits of agreement between 2 observers for a refined scoring method for nest-building behavior; 2) the effect of repeated exposure to 15 min of isoflurane on nest-building behavior; 3) the effect of 24 h of grid-floor housing, repeated exposure to 15 min isoflurane, and daily intraperitoneal injection of 0.2 mL 0.9% isotonic saline for 3 d on burrowing behavior; and 4) the effect of exposure to grid-floor housing, isoflurane, and intraperitoneal injections on fecal corticosterone metabolites, body weight, fur status, and sucrose preference in mice. SPF C57BL/6NTac female mice (n = 27) were included in the study and were assessed first for burrowing behavior, followed by 2 wk of rest and then for nesting behavior. The refined scoring method for nest-building activity had good inter observer agreement. According to this method, a single exposure to anesthesia with isoflurane led to a decrease in nest-building activity and sucrose preference; a second exposure to anesthesia with isoflurane had no effect on nest building. Neither grid-floor housing nor repeated exposure to isoflurane anesthesia had any effect on burrowing behavior in mice. In contrast, intraperitoneal injections increased burrowing behavior. In conclusion, a refined scoring method for nest-building activity test that we developed for this study proved to be objective and sensitive to the effect of an initial exposure to anesthesia with isoflurane.