Imaging mass spectrometry (MS) analysis allows scientists the ability to obtain spatial and chemical information of analytes on a wide variety of surfaces. The ability to image biological analytes is an important tool in many areas of life science research, including: the ability to map pharmaceutical drugs in targeted tissue, to spatially determine the expression profile of specific proteins in healthy vs. diseased tissue states, and to rapidly interrogate biomolecular microarrays. However, there are several avenues for improving the imaging MS experiment for biological samples. Three significant directions this work addresses include: (1) reducing chemical noise and increasing analyte identification by developing sample preparation methodologies, (2) improving the analytical figures of merit (i.e., spatial resolution, analysis time) by implementing a spatially dynamic optical system, and (3) increasing both mass spectral resolution and ion detection sensitivity by modifying a commercial time-of-flight (TOF) MS. Firstly, sample methodology schemes presented in these studies consist of obtaining both ?top-down? and ?bottom-up? information. In that, both intact mass and peptide mass fingerprinting data can be obtained to increase protein identification. This sample methodology was optimized on protein microarrays in preparation for bio tissue analysis. Other work consists of optimizing novel sample preparation strategies for hydrated solid-supported lipid bilayer studies. Sample methods incorporating nanomaterials for laser desorption/ionization illustrate the ability to perform selective ionization of specific analytes. Specifically, our results suggest that silver nanoparticles facilitate the selective ionization of olefin containing species (e.g., steroids, vitamins). Secondly, an advanced optical design incorporating a spatially dynamic optical scheme allows for laser beam expansion, homogenization, collimation, shaping, and imaging. This spatially dynamic optical system allows user defined beam shapes, decreases analysis times associated with mechanical movement of the sample stage, and is capable of increasing the MS limits of detection by simultaneously irradiating multiple spots. Lastly, new data acquisition strategies (multiple anode detection schemes) were incorporated into a commercial time-of-flight mass spectrometer to increase both sensitivity and resolution in a matrix assisted laser desorption/ionization mass spectrometer. The utility of this technique can be applied to many different samples, where high mass spectral resolution allows for increased mass measurement accuracy.