Two-dimensional (2D) cell culturing systems have been an indispensable tool for understanding the biology of cells. Conventional 2D culture models are not only suitable to study the effect of mutagens, carcinogens, and xenobiotics but also are suitable pre-clinical models that can be employed to understand drug metabolism. However, this plausibility of 2D monolayer cultures has recently been in the limelight and has been questioned on the grounds of its ability to mimic the complexities at the tissue or organ level where multiple cell types express spatiotemporally. Other concerns like multiplexity, reproducibility, and ethical concerns associated with more complex preclinical animal models make three-dimensional cellular models (3D), like spheroids and organoids, suitable for understanding the disease biology of conditions like cancer, and neurological and cognitive disorders. The current chapter provides the readers with comprehensive details of various available methods of generating 3D-invitro models, analyses and characterization of such established model systems, and their applications in drug development.