Various acaricide-resistant strains of Rhipicephalus (Boophilus) microplus, representative of the major resistance mechanisms found in Mexico and Brazil, were exposed to spinosad using the Food and Agriculture Organization of the United Nations larval packet test and adult immersion test (AIT). Larvae of all strains tested were found to be susceptible to spinosad. Conversely, spinosad did not show toxic activity toward engorged females used in the AIT. In vitro tests against larvae, nymphs, and adults of acaricide-susceptible Amblyomma americanum and Dermacentor variabilis revealed differences in activity that were dependent on tick species and parasitic life stage. Spinosad seemed to be a viable alternative to current acaricides available for tick control in the species tested. The larval packet test should be used for future monitoring of resistance, as the AIT did not provide useful information with this chemical. The potential benefit for the use of spinosad in integrated pest management or eradication programs is discussed.