Peumus boldus Mol. (boldo) is a Chilean endemic tree species, characteristic of the region's sclerophyllous forests. Increasing demand for its leaves and bark, valued for their antioxidant properties, has contributed to declining populations of this species in its natural habitat. This decline is further exacerbated by low natural germination rates and anthropogenic pressures. To support conservation efforts, efficient mass propagation protocols are needed. This study pioneered the use of temporary immersion systems (TIS) for the in vitro propagation of boldo, successfully developing a novel mass propagation protocol. To optimize the in vitro propagation of boldo shoots using the temporary immersion system (TIS), various immersion durations were tested. While a 3 min immersion resulted in a high proliferation rate (10.8), it also induced shoot vitrification, a detrimental physiological disorder. However, reducing the immersion duration to 2 min successfully prevented vitrification while still achieving a satisfactory multiplication rate of 7.2. The shoots reached an average length of 6.1 to 6.4 cm with 6-benzylaminopurine (4.44 mu M). Ex vitro rooting was achieved in 75.4% of shoots treated with 1476 mu M indole-3-butyric acid (IBA) across all tested substrates. The plantlets subsequently acclimatized with a survival rate between 77.1% and 83.3%.