Crinum malabaricum (Family: Amaryllidaceae) is a critically endangered aquatic medicinal plant endemic to India. This species is a promising natural source of bioactive compounds including galanthamine (GAL), an anti-Alzheimer drug. In vitro regeneration in the Amaryllidaceae is often challenging. This study assessed the use of meta-Topolin (mT) on in vitro regeneration of C. malabaricum. Shoot explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5, 2.5, 5.0, 7.5 and 10.0 µM mT for six weeks, whereby 7.5 µM mT resulted in the maximum multiplication of adventitious shoots, much higher than the control. The biochemical accumulation of eleven different phenolic acids was quantified by UHPLC-MS/MS analysis, and it appeared that mT-treated cultures exhibited the highest concentration of phenolic acids. In particular, increased concentrations of gallic acid, protocatechuic acid, syringic acid, p-hydroxybenzoic acid, salicylic acid and vanillic acid were detected compared to the control. mT (2.5 and 5.0 µM) produced the maximum amount of chlorogenic acid, ferulic acid, p-coumaric acid and sinapic acid. However, an increased content of caffeic acid was produced on PGR-free medium. These findings highlight the beneficial effect and validate the rising importance of mT for in vitro regeneration studies. This study will serve as a potential protocol to conserve and restore the medically important C. malabaricum.