Objective: To determine the phytochemical content, antioxidant activity in vitro and cytotoxicity of crude ethanol extract (CEE), n-hexane fraction (NHF), petroleum ether fraction (PEF), chloroform fraction (CLF) and ethyl acetate fraction (EAF) of aerial parts of Chuquiraga spinosa (C. spinosa) Lessing. Methods: Phytochemical screening was developed by color and precipitated formation. The evaluation of antioxidant activity was assessed using hydroxyl and nitric oxide radical. Total phenolic content (TPC) and total flavonoids content (TFC) were measured by using standard methods by spectrophotometry. The cytotoxic effect was determined on human tumor cell lines including MCF-7, H-460, HT-29, M-14, HUTU-80, K-562 and DU-145. Results: Phytochemical analysis confirmed the presence of phenols, flavonoids in crude extract and its all fractions. The CEE showed the highest antioxidant activity, for OH and NO radical scavenging tests (IC50 = 15.16 ± 3.45 μg/mL and IC50 = 18.91 ± 1.13 μg/mL, respectively). TPC was found to be the highest in the CEE (121.36 mg of gallic acid equivalent/g of dried extract) compared to other fractions. The ranking order of NHF, PEF, CLF, EAF and CEE for TFC was 21.17 < 35.20 < 62.19 < 70.25 < 78.25 mg quercetin equivalent/g of dried extract. The crude ethanolic extract (μg/mL) showed a high cytotoxicity on MCF-7 (IC50 = 9.25 ± 0.81), K-562 (IC50 = 7.34 ± 1.00), HT-29 (IC50 = 8.52 ± 2.69), H-460 (IC50 = 5.32 ± 1.05), M-14 (IC50 = 8.30 ± 0.60), DU-145 (IC50 = 7.09 ± 0.09), HUTU-80 (IC50 = 6.20 ± 0.50). Conclusions: The study showed that CEE of the aerial parts of C. spinosa can be measured as a natural source of antioxidant which might be effective towards preventing or slowing oxidative stress related to chronic diseases as well as cytotoxic agent.