Cell behavior is significantly different in two-dimensional and three-dimensional culture conditions, and a number of methods have been developed to establish and study three-dimensional cellular arrays in vitro. When grown under nonadherent conditions, many types of cells form structures called multicellular spheroids (MCSs), which have been popular models to study cell behavior in a three-dimensional environment. The histoarchitecture of MCSs derived from malignant cells resembles that of tumors, and there is rapidly increasing interest in using these structures to more accurately understand the dynamics of cancer cells in situ, including their responses to chemotherapeutics. Confocal microscopy is an extremely useful method to investigate cell behavior in MCSs due to its ability to more clearly image fluorescent probes at some depth in three-dimensional structures. This chapter describes some basic approaches toward visualizing a variety of fluorescent probes in MCSs.