Researchers are recognizing the limitations of two-dimensional (2D) cell cultures, given the fact that they do not reproduce the morphology and biochemical features that the cells possess in the original tissue. As an alternative, the three-dimensional (3D) cell culture approach offers researchers the possibility to study cell growth and differentiation under conditions that more closely resemble the in vivo situation with regard to cell shape and cellular environment. Currently, 3D culture models are being employed in many areas of biomedical research because they offer a more realistic milieu than 2D cultures. The era of 2D culture techniques is moving towards a new epoch of culture systems in 3D. The present review is focused on topics of research on 3D cell cultures in virology and their use in antiviral drug development.