The Amazon rainforest is an important reservoir of biodiversity, offering vast potential for the discovery of new bioactive compounds from plants. In vitro studies allow for the investigation of biological processes and interventions in a controlled manner, making them fundamental for pharmacological and biotechnological research. These approaches are faster and less costly than in vivo studies, providing standardized conditions that enhance the reproducibility and precision of data. However, in vitro methods have limitations, including the inability to fully replicate the complexity of a living organism and the absence of a complete physiological context. Translating results to in vivo models is not always straightforward, due to differences in pharmacokinetics and biological interactions. In this context, the aim of this literature review is to assess the advantages and disadvantages of in vitro approaches in the search for new drugs from the Amazon, identifying the challenges and limitations associated with these methods and comparing them with in vivo testing. Thus, bioprospecting in the Amazon involves evaluating plant extracts through bioassays to investigate pharmacological, antimicrobial, and anticancer activities. Phenolic compounds and terpenes are frequently identified as the main bioactive agents, exhibiting antioxidant, anti-inflammatory, and antineoplastic activities. Chemical characterization, molecular modifications, and the development of delivery systems, such as nanoparticles, are highlighted to improve therapeutic efficacy. Therefore, the Amazon rainforest offers great potential for the discovery of new drugs; however, significant challenges, such as the standardization of extraction methods and the need for in vivo studies and clinical trials, must be overcome for these compounds to become viable medications.