Natural deep eutectic solvents have a high solvation and food-grade characteristics, which make them a preffered choice for extracting bioactives to develop nutraceutical and cosmetic formulations. This research aimed to investigate the systematic effects of several NADESs on the ultrasonic-assisted extraction of steroidal saponins from the rhizomes of Trillium govanianum Wall. ex D.Don, which is utilized in traditional Ayurvedic medicine. After screening of fifteen different NADESs, choline chloride: lactic acid (1:1) (NADES 1) was found most effective eutectic solvent for the extraction of steroidal saponins performed by the three-round micro-extraction (1 mL). Compared with hydro-ethanolic solvent-based extraction, lactic acid-derived NADESs showed higher extractability of steroidal saponins. The extraction efficiency of bioactive steroidal saponins e.g. borassoside E and protodioscin was also enhanced with NADES 1. The effects of extraction variables such as S:L ratio, working duration, and temperature on the extraction of steroidal saponins by NADES 1 was also studied and optimized.Furthermore, optimized parameters were validated by performing 100 mL NADES 1-based extraction to recover phytomolecules and NADES 1 by using Diaion HP20 resin column chromatography. As a proof of concept, this study suggested that lactic acid-derived NADESs are effective media for the extraction of steroidal saponins from biomass. The cytocompatibility assay of NADESs with NRK-52E and IEC-6 cells revealed the safe nature of NADESs to normal animal cells. The steroidal saponins-enriched extract obtained with NADES 1 has shown promising antagonistic effects on acetylcholinesterase activity.