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Growing worldwide, the genus Ephedra (family Ephedraceae) had a medicinal, ecological, and economic value. The extraordinary morphological diversity suggests that Ephedra was survivor of an ancient group, and its antiquity is also supported by fossil data. It has recently been suggested that Ephedra appeared 8–32 million years ago, and a few megafossils document its presence in the Early Cretaceous. Recently, the high analytical power provided by the new mass spectrometry (MS) instruments is making the characterization of Ephedra metabolites more feasible, such as ephedrine series. In this regard, the chemical compounds isolated from crude extracts, fractions, and few isolated compounds of Ephedra species were characterized by MS-based techniques (LC-MS, LC-ESI-MS, HPLC-PDA-ESI/MS, LC-DAD-ESI/MSn, LC/Orbitrap MS, etc.). Moreover, we carry out an exhaustive review of the scientific literature on biomedicine and pharmacotherapy (anticancer, antiproliferative, anti-inflammatory, antidiabetic, antihyperlipidemic, antiarthritic, and anti-influenza activities; proapoptotic and cytotoxic potential; and so on). Equally, antimicrobial and antioxidant activities were discussed. This review is focused on all these topics, along with current studies published in the last 5 years (2015–2019) providing in-depth information for readers.