A flea larval bioassay was developed by an international team of scientists to monitor the susceptibility of fleas (Ctenocephalides felis) to imidacloprid (Advantage, Bayer HealthCare). The assay was validated using laboratory and field isolates of C. felis. Flea eggs representing different field isolates of C. felis were collected by veterinarians in the United States, United Kingdom, and Germany. Of the 972 flea isolates obtained during the 5-year study, 768 contained sufficient numbers of eggs to conduct the larval bioassay. Greater than 5% survival occurred for only six of the field isolates evaluated. Further evaluation and analysis of these isolates demonstrated that they did not differ significantly in their susceptibility to imidacloprid from the reference strains used to develop the assay. Collections of field flea isolates will continue in an attempt to detect and document any change in the susceptibility of field flea populations to imidacloprid.