<p>Spinach (<i>Spinacia oleracea</i>), a highly valuable leafy vegetable, presents significant challenges for <i>in vitro</i> regeneration due to its recalcitrant nature, hindering genetic improvement efforts. This study aimed to develop an efficient regeneration protocol for spinach by investigating the effects of novel small molecules in tissue culture media. In addition to traditional plant growth regulators, we incorporated a cytokinin oxidase inhibitor (3TFM-2HE) and chlorpromazine (CPZ), a combination that has not been previously reported for plant tissue culture. Various combinations and concentrations of these regulators were evaluated for direct and indirect organogenesis. While direct organogenesis proved unsuccessful, a two-step protocol involving callus induction followed by shoot regeneration yielded positive results. To initiate the process, competent callus formation was induced through a combination of 10 µM Naphthaleneacetic Acid (NAA), 10 µM 2-isopentenyladenosine (2iPR), 1 µM 3TFM-2HE, and 50 µM CPZ. Subsequently, the introduction of 5 µM Gibberellic Acid (GA₃) stimulated successful shoot regeneration. This is the first <i>in vitro</i> regeneration report for the commercially important spinach cultivar <i>Palco</i>, with no prior tissue culture studies. This protocol provides a valuable tool for spinach improvement and demonstrates the potential of cytokinin oxidase inhibitors and CPZ for enhancing regeneration in other recalcitrant species.</p> Graphical abstract <p></p>