The current investigation utilized stem nodes from pre-established aseptic lines of Mussaenda pubescens as explants to optimize an efficient in vitro propagation protocol and investigated the effect of different light qualities (white, red, blue, and green) on metabolite accumulation in micropropagated plantlets. The findings demonstrated that the optimal medium for shoot proliferation was Murashige and Skoog basal medium supplemented with 6-Benzylaminopurine 2.0 mg·L⁻¹ and α-naphthaleneacetic acid 0.2 mg·L⁻¹, achieving a multiplication coefficient of 12.2 after 30 days. Rooting was more effective on Murashige and Skoog basal medium containing α-naphthaleneacetic acid 0.1 mg·L⁻¹ and activated charcoal 1 g·L⁻¹, resulting in a 100% rooting rate. During acclimatization, a substrate mixture of perlite:vermiculite: peat soil (1:1:1) promoted vigorous root development with a 100% survival rate at post-transplantation. Light quality significantly influenced plant morphology: red light stimulated stem elongation, while blue light increased biomass accumulation. Broad-target metabolomics revealed distinct metabolite profiles under different light spectra, with differentially accumulated metabolites primarily belonging to terpenoids, organic acids, lipids, and flavonoids. Specifically, red light enhanced the levels of terpenoids and lipids; blue light promoted the synthesis of specific triterpenoid saponins and lipids; while green light increased the content of certain terpenes and broadly upregulated a wide spectrum of lipids. This work provides a robust framework for the commercial micropropagation of Mussaenda pubescens and elucidates the strategic use of light quality to enhance the production of its valuable medicinal metabolites, including terpenoids and lipids.