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Abstract Cancer is a class of diseases characterized by uncontrolled cell growth. One of the main aims of developing new therapies is to use natural resources to induce apoptosis. LC-ms/ms analysis of a methanolic extract of Ephedra alata (E.A.) allowed the identification of 20 secondary metabolites, including flavonoids, phenolic acids, and proanthocyanidins. Antiproliferative effect was assessed by crystal violet assay. Antimigration effect was tested by wound healing assay and apoptosis induction was determined by annexin binding assays, Hoechst staining, ROS production, and activation of apoptotic proteins. The results indicated that exposure of breast cancer cells to E.A. extract significantly reduced cell viability in a dose and time-dependent manner and inhibited the migration of 4T1 cells at a low dose. Moreover, treatment of cells with E.A. extract induced apoptosis, as it was detected by Annexin V/7 AAD, Hoechst staining, ROS production, and the activation of caspases. Abbreviation: BSA bovine serum albumin DMSO dimethyl sulfoxide EDTA ethylenediaminetetraacetic acid LC-ms/ms liquid chromatography-mass spectrometry NAC N-acetyl-l-cysteine PARP poly(ADP-ribose) polymerase PMSF phenylmethylsulfonyl fluoride RIPA radioimmunoprecipitation assay buffer ROS reactive oxygen species RPMI Roswell park memorial institute SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.