This study aimed to investigate the chemical compositions of Tripterygium hypoglaucum roots and their neuroprotective effects. The ethyl acetate fraction of the ethanol extract from T. hypoglaucum roots was isolated and purified using the techniques of silica gel column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative high-performance liquid chromatography (HPLC). The structures of the compounds were elucidated through high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, and computational electron circular dichroism (ECD) analysis. The neuroprotective effects of the compounds were assessed by lipopolysaccharide (LPS)-induced mouse microglial cells (BV2 cells). A total of 13 compounds were successfully isolated and identified from T. hypoglaucum roots, including (+)-catechin (1), (−)-epicatechin (2), (−)-epicatechin (3), 3'-O-methylcatechin (4), 2S,3S-3,5,7,3'-tetrahydroxy-5'-methoxyflavane (5), (−)-afzelechin (6), (−)-3-O-acetylcatechin (7), (+)-taxifolin (8), dihydrokaempferol (9), luteolin (10), quercetin (11), 8R-3-hydroxy-1-(2-hydroxy-5-methoxyphenyl)-2-(4-hydroxy-3-methoxyphenyl)-1-propanone (12), 8R-evofolin B (13) in which seven catechins and their derivatives (1-7), four flavanol compounds (8-11), and two diphenylpropane derivatives (12-13) were included. Notably, compounds 3, 7, 8, 12, and 13 were isolated from T. hypoglaucum for the first time. Compounds 1, 2, 4, 6–8, and 11 exhibited substantial inhibitory effects on nitric oxide (NO) production in LPS-induced BV2 cells. The IC₅₀ value of compound 4 was determined to be 12.89 ± 3.89 µmol/L, which is comparable to that of the positive control drug minocycline (IC₅₀ = 16.26 ± 4.97 µmol/L). It demonstrated that T. hypoglaucum roots were rich in flavonoids and diphenylpropane compounds in which flavonoids could exhibit neuroprotective effects on LPS-induced BV2 cell model by suppressing nitric oxide production.