Background <p>Hookworm infections such as <i>Ancylostoma braziliense</i> and <i>A. ceylanicum</i> pose a significant threat to pets and are implicated in causing zoonotic diseases. Despite the availability of preventatives, compliance can be lacking. Increasing pet owner options and combining endo- and ectoparasite treatments might improve this compliance. In four separate studies, we investigated the efficacy of Simparica Trio® (Zoetis Inc., Parsippany, NJ, USA) containing minimum dosages of 1.2&#xa0;mg/kg sarolaner, 24&#xa0;µg/kg moxidectin and 5&#xa0;mg/kg pyrantel against fourth- and fifth-stage larvae (L₄ and L₅, respectively) and adults of <i>A. braziliense</i> and adults of <i>A</i>. <i>ceylanicum</i> in dogs.</p> Methods <p>Four negatively controlled, randomised and blinded laboratory studies were conducted against induced infections of <i>A. braziliense</i> and <i>A. ceylanicum</i>, with the interval between infection and treatment selected to evaluate efficacy against each targeted stage. Each treated dog received a single oral dose of Simparica Trio® at the recommended label dose. Necropsy was conducted for worm recovery on day 7 or 8 post-treatment.</p> Results <p>No treatment-related adverse events were recorded in any of the studies. No worms were recovered from any of the Simparica Trio®-treated dogs in all four studies, thus resulting in 100% efficacy (<i>P</i> ≤ 0.0005) of Simparica Trio® against all stages of <i>A. braziliense</i> and the adult stage of <i>A. ceylanicum</i> in dogs.</p> Conclusions <p>Simparica Trio® containing sarolaner, moxidectin and pyrantel was highly effective against induced infections of L₄, L₅ and adult stages of <i>A. braziliense</i> and the adult stage of <i>A. ceylanicum</i> in dogs.</p> Graphical Abstract <p></p>