Tissue engineering and regenerative medicine aim to substitute failing structural, secretory, or immunomodulatory functions of human tissues and organs, which opens new perspectives to overcome chronic diseases. Based on tissue engineering principles in combination with accelerated progress in fabrication strategies, advanced in vitro models such as three-dimensional multicellular tissue equivalents and microfluidic organ-on-chip systems have gained increasing relevance for applications in personalized medicine, as well as drug development and testing. Novel marker-independent, non-, or minimally invasive in situ monitoring technologies that are combined with tissue engineering and personalized medicine strategies allow a better understanding of comprehensive regenerative processes and have the potential to advance clinical translation. Raman imaging is a spectroscopy-based approach, first described about 100 years ago as the Raman effect, caused by vibrational interactions between the molecules of a sample and the photons of a light source. The resulting spectrum is molecule-specific and provides insights on the (bio-)chemical composition of a sample, going beyond the sensitivity of other image-based analytical or diagnostic tools. The insensitivity to water promoted the implementation of Raman spectroscopy as an ideal tool to investigate biological samples in situ, boosting its application in the field of biomedicine over the last decades. Furthermore, improvements in the development of endoscopic Raman setups contribute to the realization of Raman-based clinical, in vivo imaging. This chapter aims to introduce Raman imaging, including the basics on the physical principle and technical prerequisites, and highlights advances in applications of Raman techniques in tissue engineering and regenerative medicine.