Lepidium meyenii (Maca), originated from Peru, has been cultivated widely in China as a popular health care food. However, the chemical and effective studies of Maca were less in-depth, which restricted its application seriously. To ensure the quality of Maca, a feasible and accurate strategy was established. One hundred and sixty compounds including 30 reference standards were identified in 6 fractions of methanol extract of Maca by UHPLC-ESI-Orbitrap MS. Among them, 15 representative active compounds were simultaneously determined in 17 samples by UHPLC-ESI-QqQ MS. The results suggested that Maca from Yunnan province was the potential substitute for the one from Peru. Meanwhile, the neuroprotective effects of Maca were investigated. Three fractions and two pure compounds showed strong activities in the 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced zebrafish model. Among them, 80% methanol elution fraction (Fr-5) showed significant neuroprotective activity, followed by 100% part (Fr-6). The inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was a possible mechanism of its neuroprotective effect.