Kaempferia galanga L. (Zingiberaceae) is a commercially and therapeutically vital endangered medicinal plant whose natural populations are rapidly depleting due to over harvesting and sluggish conventional propagation methods. This study aimed to establish a highly efficient, reproducible in vitro propagation protocol using rhizome bud explants, alongside a comparative qualitative phytochemical profiling of in vitro regenerated versus naturally grown plants. Explants were cultured on Murashige and Skoog (MS) basal medium supplemented with varying concentrations and combinations of 6-benzyl aminopurine (BAP), Kinetin (Kn), α-Naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) and Indole-3-acetic acid (IAA). The quantitative evaluation of plant growth regulator combinations revealed that the maximum multiple shoot buds (MSBs) induction frequency (96%) and the highest number of shoots per explant (4.53 ± 0.25) were recorded on MS medium supplemented with 1.5 mg/l BAP and 1.0 mg/l NAA within 15-23 days of culture. For shoot elongation, MS medium fortified with 2.0 mg/l BAP and 0.5 mg/l Kn exhibited the optimum response, expanding initial 2.16 ± 0.07 cm buds to a maximum length of 4.54 ± 0.04 cm. Superior rhizogenesis (90% rooting frequency; 4.25 ± 0.10 roots/shoot; 3.65 ± 0.07 cm root length) was achieved on full strength MS medium supplemented synergistically with 0.5 mg/l IBA within 14-17 days. Hardened seedlings demonstrated an 80% survival rate upon successful acclimatization to ex vitro conditions. Comparative qualitative phytochemical screening for secondary metabolites revealed that in vitro developed plantlets possessed elevated levels of specific alkaloids (responding intensely to Hager's, Mayer's and Tannic acid reagents) and coumarin (+++) compared to naturally grown plants (+), whereas saponins were absent in the tissue cultured clones. These findings substantiate that the optimized micropropagation protocol not only facilitates rapid mass scale conservation but also enhances the elicitation of highly valued pharmaceutical bioactive compounds, offering a sustainable alternative for commercial therapeutic exploitation.