Advances in two-dimensional (2D) and three-dimensional (3D) cell culture over the last 10 years have led to the development of a plethora of methods for cultivating tumor models. More recently, cellular co-cultures have become a suitable testbed. The first portion of this review focuses on co-culturing methods that have been developed in recent years utilizing the multicellular tumor spheroid model. The latter portion describes techniques that are used to analyze the proteomes of mono-or co-cultured tumor models, with a focus on mass spectrometry (MS)-based analyses. Protein profiles are important indicators of the tumor heterogeneity. Therefore, there is a specific focus within this review on analysis by MS and MS imaging methods evaluating the proteomic profiles of 2D and 3D co-cultures. While these models are incredibly important for biological research, so far, they have not been widely explored on the proteomic level. With this review, we aim to introduce these systems to an analytical audience, with the goal of highlighting MS as an underutilized tool for proteomic analysis of tumor models.