Background: Although the use of animals in various experimental activities is still necessary for the advancement of science, research for the development, validation, and recognition of new microchip technologies has increased in recent years, which characterizes a great advance for science. Alternative biotechnological methods aim to replace the use of animal models in scientific experiments or reduce their use when replacement is not possible, which corroborates the principle of the Replacement, Reduction, and Refinement (3Rs)— total replacement, reduction, refinement. Thereby, it is possible to overcome inaccurate predictions caused by distinct elements between species, significantly reduce research costs and time by promoting a comprehensive scientific advance in a more favorable regulatory environment. This review aims to highlight current and future perspectives of tissue bioengineering aimed at possible alternative or substitute methods to animal models in preclinical research in biomedical applications. Methods: The search strategy was based on the PubMed/Medline, SciELO, and Web of Science databases, as well as books on the respective topics, accessed in the period between October 2024 to March 2025. The keywords were “alternative biotechnologies”, “non animals”, “tissue bioengineering”, “organ on chip”, “biomaterials”, “tissue”, “engineering”, “bone” from the last five years, totalizing 4429 articles. The eligibility criteria, established as cutoff points, were described in the methodology. Conclusions: Thus, new tissue bioengineering tools and technologies are rapidly emerging to innovate in vitro practices. Examples include organs-on-a-chip and 3D biomimetic structures with tissue-like dynamic properties to enable in-depth investigation of disease mechanisms.