Chagas disease is caused by the parasite Trypanosoma cruzi and affects millions of people worldwide, having no effective cure. The main sanitary emergency is related to patients with chronic infection, which accumulate comorbidities causing patient death. However, actual chemotherapeutic treatments do not effectively address the chronic forms of the disease. Invertebrates are a relevant source of antimicrobial peptides (AMPs) as part of the innate immune system for their protection. The AMP M-PONTX-Dq3a, isolated from the Dinoponera quadriceps ant venom, has shown very effective antimicrobial and trypanocidal activities. Although M-PONTX-Dq3a has better activity that the current therapies, the peptide length has limited its possibilities to reach clinical application. In this investigation, we aimed to dissect the trypanocidal effect of M-PONTX-Dq3a fragments and to study the activity of substituted analogs, to improve not only peptide trypanocidal activity and bioavailability, but also production costs. Our studies have led to the identification of two smaller peptides, M-PONTX-Dq3a [1-15] and [Lys]<sup>3</sup>-M-PONTX-Dq3a [3-153-15 with similar trypanocidal activities that the parent peptide has against the three forms of T. cruzi benznidazole-resistant Y strain. Both peptides represent promising candidates to develop novel and effective trypanocidal bio-therapeutic agents, opening new avenues for the treatment of chronic patients.