The ability to visualize biochemical interactions between microbial communities using MALDI MSI has provided tremendous insights into a variety of biological fields. Matrix application using a sieve proved to be incredibly useful, but it has many limitations that include uneven matrix coverage and limitation in the types of matrices that could be employed in studies. Recently, there has been a concerted effort to improve matrix application for studying agar plated microbial cultures, many of which utilized automated matrix sprayers. Here, we describe the usefulness of using a robotic sprayer for matrix application. The robotic sprayer has two-dimensional control over where matrix is applied, and a heated capillary that allows for rapid drying of the applied matrix. This method provided a significant increase in MALDI sensitivity over the sieve method, as demonstrated by FT-ICR MS analysis, facilitating the ability to gain higher lateral resolution MS images of Bacillus subtilis than previously reported. This method also allowed for the use of different matrices to be applied to the culture surfaces.