As essential oils (EOs) represent a new source of efficient and safe agents for health nowadays, the present review brings together the in vitro methods widely used to evaluate the antioxidant and some biological activities especially, antidiabetic, anticancer, antimicrobial, and anti-inflammatory activities of EOs, in order to valorize these EOs and to highlight their potential benefits. Moreover, each method cited is along with its aim, principle, advantages and limitations, experimental protocols, and notes. Hence, this review will help researchers working on EOs, to save time while accessing this summary document on the one hand, and on the other hand, it will contribute to scientific approval of in vitro antioxidant and biological effects of EOs for future useful purposes.