The American Cancer Society estimates that there will be more than 1.7 million new cases of cancer diagnosed in 2018, so the need for understanding the molecular mechanisms of cancer is great. Research has long suggested that the necrotic core of tumors may not receive the full dose of treatment and the cells residing there can ultimately be responsible for cancer progression.Spatial analysis of chemical species can allow one to detect small changes over time and differences between data sets. This type of analysis allows for important questions to be critically analyzed beyond the scope of a simple one-dimensional analysis. These spatial localizations can be analyzed in numerous ways. However, the techniques utilized within this document include Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI MSI) and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) followed by liquid chromatography tandem mass spectrometry (LC-MS/MS).Tumors in vivo have a complex microenvironment and researchers have spent decades developing different models to test the efficacy of novel treatments. The work described in this document utilizes a three-dimensional cell culture system. These multicellular aggregates referred to as tumor spheroids, display pathophysiological gradients that are more complex than conventional two-dimensional cell cultures in addition to being higher throughput than animal models.The main focus of this work is liposomal drug carrier distribution within tumor spheroids. These nano-carriers have the ability to positively alter pharmacokinetics profiles and have the capability for encapsulation of therapeutics. Doxorubicin was the chemotherapeutic utilized in this work due to its native fluorescent properties as well as its ability to be detected using mass spectrometry. The work described herein develops a new platform that can readily be analyzed using MALDI MSI, something that has not previously been done. Additional work presented herein describes a SILAC labeling approach that creates a "zip code" system for proteins residing in the various cellular populations of a tumor spheroid.All together this work focuses on the spatial localizations of endogenous and exogenous molecules within tumor spheroids.