Introduction: Drugs resistant Plasmodium falciparum is a recurring issue that threatens public health. New anti-plasmodial drugs are needed to overcome this problem. The aim of this study was to characterize in vitro an-tiplasmodial activity of Withania frutescens (Solanaceae) against chloroquine-resistant Plasmodium falciparum strain. Methods: The in vitro antiplasmodial activity of leaves and roots extracts from Withania frutescens was performed in 96 well plates and preliminary phytochemical analysis was performed for the active fractions. The toxicity of the plant extract against CHO (Chinese Hamster Ovary) cells was assessed using the tetrazolium salt MTT col-orimetric assay. The selectivity index (SI) was calculated as the ratio between the cytotoxic and antiparasitic activities. Results: The methanol extract of W. frutescens leaves showed a good antiplasmodial activity (CC 50 18.1 μg/ml). Furthermore, ethyl acetate and butanol fractions showed promising in vitro antiplasmodial activity with a se-lectivity index of 3.1 and 2.4, respectively. The roots of W. frutescens were found to be inactive with a CC 50 value > 80 μg/ml. Conclusion: The antiplasmodial activity of W. frutescens may in part be attributed to the presence of polyphenolic and flavonoid compounds. Based on our results, ethyl acetate and butanol leaves fractions could be considered as a promising source for the development of putative antiplasmodial drugs.