Traditionally, velvet antler (VA) has been used as a medicine or dietary supplement in East Asia. It contains biologically active compounds that exert anti-inflammatory, anti-fatigue, anti-aging, and anticancer effects. Although demand for VA has increased globally, its supply and consumption are limited due to the low recovery of its bioactive compounds from traditional decoctions. Therefore, alternative extraction methods are required to enrich the active compounds and enhance their biological efficacy. The extract has been reported to protect against neuropathological conditions in brain cells and suppress oxidative stress and neuroinflammation-crucial for the initiation or progression of neurodegenerative diseases. Therefore, VA is a potential therapeutic agent for neurodegenerative diseases. However, the beneficial effects of VA on astrocytes, which are the predominant glial cells in the brain, remain unclear. In the present study, we investigated the protective effects of enzymatically digested VA extract (YC-1101) on the mitochondria in astrocytes, which are essential organelles regulating oxidative stress. Proteomic and metabolomic results using liquid chromatography-mass spectrometry (LC-MS/MS) identified enriched bioactive ingredients in YC-1101 compared to hot water extract of VA. YC-1101 displayed significant protective effects against mitochondrial stressors in astrocytes compared with other health functional ingredients. Altogether, our results showed improved bioactive efficacy of YC-1101 and its protective role against mitochondrial stressors in astrocytes.