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Five studies (two dose determination, two dose confirmation, and one speed of flea kill study) were conducted to assess the immediate (therapeutic) efficacy and long-term persistent (preventive) efficacy of a single spot-on application containing the novel acaricide and insecticide tigolaner in combination with emodepside and praziquantel (Felpreva®, Vetoquinol S.A. Lure, France) applied to cats artificially infested with Ctenocephalides felis. Eight cats per group were randomly allocated to 0, 1×, 1.3× and 2× of the minimum dose (14.5 ​mg/kg body weight) of tigolaner (dose determination studies) or randomly allocated to 0 and 1× of the dosage (dose confirmation studies). Onset of efficacy was assessed in a speed of flea kill study on an existing flea infestation 8, 12 and 24 ​h after treatment and reassessed after monthly flea reinfestation until 13 weeks post-treatment. Efficacy was calculated according to the Abbott formula using arithmetic means. Efficacy was claimed when (i) control groups were adequately infested (flea retention ≥ 50%) at each time-point in the studies; (ii) flea counts in treated groups were significantly lower (P ​≤ ​0.05) than flea counts in control groups; and (iii) calculated efficacy was ≥ 90% (speed of flea kill study) and ≥ 95% (dose determination and dose confirmation studies). Tigolaner at 14.5 ​mg/kg body weight was 100% effective against fleas on Day 1 (immediate, therapeutic efficacy) in both, dose determination and dose confirmation studies. The long-term persistent efficacy in week 13 ranged between 96.3% and 100%. Fleas were rapidly killed within 12 ​h after treatment (100% flea reduction, immediate efficacy). New flea infestations were successfully prevented for 8 weeks (98.9–100% flea reduction) within 8 ​h after reinfestation, and at week 13 (96.3% flea reduction) within 24 ​h after reinfestation.