Background: The black-legged (or deer) tick, Ixodes scapularis, commonly infests dogs in the USA and is the vector of important zoonotic pathogens, including Borrelia burgdorferi, the causative agent of Lyme disease. Rapid onset of activity is important in reducing the feeding activity of ticks, thereby reducing the possibility of transmission of infections. The speed of kill of a novel oral combination product, Simparica Trio ™ containing sarolaner, moxidectin and pyrantel was evaluated in a well-controlled laboratory study against an existing infestation and subsequent weekly induced infestations of I. scapularis ticks on dogs. Methods: Dogs were allocated randomly based on host suitability tick counts to treatment with a single dose of either placebo or Simparica Trio ™ at the minimum label dose of 1.2 mg/kg sarolaner, 24 µg/kg moxidectin and 5 mg/ kg pyrantel (as pamoate salt). All dogs were infested with approximately 50 unfed adult I. scapularis ticks at a 1:1 sex ratio on Days −2, 7, 14, 21, 28 and 35. Tick counts were conducted at 8, 12 and 24 h after treatment on Day 0 and after each subsequent infestation. Results: No treatment-related adverse events occurred during the study. Dogs in the placebo-treated group maintained adequate tick infestations for the duration of the study. Day 0 tick counts at 8 h after treatment with Simparica Trio ™ were reduced relative to placebo against an existing infestation with efficacy of 67.5%, demonstrating that Simparica Trio ™ started killing ticks soon after treatment. Efficacy was 98.4 % at 12 h and 99.4% at 24 h. Rapid speed of kill was maintained throughout the month, with efficacy of ≥ 94.2% at 24 h after re-infestation through Day 28. Conclusions: A single dose of Simparica Trio ™ administered orally to dogs at the minimum label dose of 1.2 mg/kg sarolaner, 24 µg/kg moxidectin and 5 mg/kg pyrantel (as pamoate salt) was safe and began to kill existing I. scapularis ticks within 8 h after treatment and resulted in ≥ 94.2% efficacy within 24 h against re-infestations for a month.