Recent studies from drug screening and disease progression have shown that 3D cell culture can better represent the in vivo or clinical condition compared to 2D monolayer culture. However, due to the limited optical transparency, characterization of the live 3D cell culture model is often challenging and fails to extract further information this culture system can provide. In this lecture, the recent development of shear wave elasticity imaging (SWEI) and photoacoustic (PA) imaging to study 3D culture systems will be covered. A 3D optical resolution PA microscope (OR-PAM) along with molecular labeling has been developed to study cell movement and cell-cell interactions. In addition, novel ultrasound- and optics-based shear wave imaging methods have been used to non-invasively quantify the matrix stiffness in 3D culture condition. Both techniques were developed to demonstrate the correlation between matrix stiffness and T cell interstitial migration during immunotherapy. The goal of this lecture is to present the influential role of these combinatory techniques from optics and ultrasound in in vitro research and the development of new treatment strategies.