<p><i>Acmella oleracea</i>, commonly called toothache plant, is a multipurpose taxon with wide-ranging uses, from culinary to traditional medicine. Availability of this plant is highly limiting at its industrial scale. However, the crop has a high potential for large-scale in vitro multiplication and genetic transformation. Optimisation of de novo plant regeneration and a high rate of shoot multiplication is the first step in plant transformation studies. Here, we present a method for optimizing callus formation and de novo shoot regeneration from <i>A. oleracea</i> leaf explants using response surface methodology. This method can predict the type, concentration, and combination of growth regulators required to elicit different responses in explants. It was used to optimize and predict combinations of auxins (NAA, IAA, 2, 4-D) with cytokinins (BAP, TDZ, Kn) to induce callus formation and shoot regeneration from leaf explants of <i>A. oleracea</i>. The main findings of this study are: (1) Auxins and cytokinins, when tested individually, could not induce any differentiation in the cultured plant tissues, (2) A combination of auxins and cytokinin is required for induction of callus and subsequently the shoot regeneration, (3) A combination involving IAA was more efficient than NAA in inducing shoot regeneration, (4) Induction of maximum percentage of shoot regeneration (90%) with a high shoot intensity (2.8 shoots/explant) occurred on MS medium containing 1&#xa0;mg/L of IAA and 3&#xa0;mg/L of BAP. This research highlights the effectiveness of response surface methodology in optimizing the tissue culture system of <i>Acmella oleracea</i>, laying a foundation for future plant transformation studies.</p>