This chapter explains the importance of 3D cell culture and highlights its potential and benefits in comparison to traditional 2D cultivation. Since 3D cell culture is supposed to mimic the in vivo situation, we will also have a close look on the complex composition of the in vivo microenvironment and why 2D cultivation does not resemble the in vivo situation. Furthermore, we will cover the most important approaches for 3D cell culture which are matrix-free and matrix-based cultivation as well as bioprinting. Also, the main applications of 3D cell culture, in vitro tissue or disease models and tissue engineered constructs for tissue repair or regeneration, will be introduced. Finally, you will learn that 3D cell culture has its limitations and challenges when it comes to the analysis and monitoring of 3D cell culture processes.