Tumor-targeted drug delivery systems are promising for their advantages in enhanced tumor accumulation and reduced toxicity towards normal organs. However, few nanomedicines have been successfully translated into clinical application. One reason is the gap between current pre-clinical and clinical studies. The prevalent in vitro models utilized in pre-clinical phase are mainly based on the two-dimensional (2D) cell culture and are limited by the difficulty of simulating three-dimensional physiological conditions in human body, such as three-dimensional (3D) architecture, cell heterogeneity, nutrient gradients and the interaction between cells and the extracellular matrix (ECM). In addition, traditional animal models have drawbacks such as high-cost, long periods and physiological differences between animal and human. On the other hand, the employment of 3D tumor cell culture models, especially multicellular tumor spheroids (MCTS), has increased significantly in recent decades. These models have been shown to simulate 3D structures of tumors in vitro with relatively low cost and simple protocols. Currently, MCTS have also been widely exploited in drug delivery system research for comprehensive study of drug efficacy, drug penetration, receptor targeting, and cell recruitment abilities. This review summarizes the delivery barriers for nano-carriers presented in tumor microenvironment, the characteristics and formation methods for applicable multicellular tumor spheroid culture models and recent studies related to their applications in tumor-targeted drug delivery system research.