Biologists handled Canis lupus rufus (Red Wolf) pups from 12 wild litters over 3 years to determine if den interference and handling negatively impacted neonatal survival. Litters were handled for blood collection and transponder placement on one of 2 den visits approximately 13 days apart when pups were approximately 5 days and 19 days old, respectively. No biologically important difference in the proportion of pups surviving was observed between subsequent visits, nor in comparison to historical data from dens where pups were not handled but rather documented based on autumn trapping surveys.