Cat fleas (Ctenocephalides felis (Bouché)) are a common flea species mostly found on cats and dogs. They not only cause discomfort to pets and their owners but also act as important vectors for human and pet zoonoses. Over the past 15 yr, the control of cat fleas on pets has been revolutionized through the use of various treatments. At present, fipronil and imidacloprid are used for flea control in Taiwan and are available in spot-on and spray formulations. Outside Taiwan, spinosad in tablet form is also available. In this study, we examined the effects of the aforementioned three insecticides on laboratory-reared and field-collected cat flea larvae. We developed a new technique for detecting flea susceptibility using a single larval bioassay with 96-well microtiter plates via contact and oral applications. Compared to the lab strain, the field strain exhibited lower susceptibility to fipronil, with the latter showing resistance levels two to four times higher than that of the former. By contrast, no difference in susceptibility was found between the two strains when tested with spinosad and imidacloprid. Our new technique was found to be stable, standardized, more efficient, convenient, and reproducible when compared to present techniques.