This research aimed to examine the impact of argumentation-based chemistry instruction on the argumentation self-efficacy of pre-service teachers' (PSTs') and their perceptions regarding the effectiveness of this instruction on their argumentation self-efficacy. This exploratory study involved a cohort of PSTs who actively participated in a series of argumentation-based chemistry activities facilitated by their classroom teacher over an 11-week period, with each activity spanning 1 or 2 weeks. The introduction of argumentation preceded the exploration of chemistry topics, including heat and temperature, gas laws, physical and chemical change, solubility, distinctive properties of matter, chemical reactions, and acids-bases. Data sources comprised the self-efficacy scale for argumentation, the evaluation of instruction for enhancing self-efficacy survey, and semi-structured interviews. The findings revealed an increase in PSTs' self-efficacy for argumentation after 11 weeks of argumentation-based chemistry instruction. These results suggest that PSTs can significantly enhance their self-efficacy for argumentation when provided with instruction tailored to incorporate argumentation principles.