Mass spectrometry imaging (MSI) is a rapidly evolving field for monitoring the spatial distribution and abundance of analytes in biological tissue sections. It allows for direct and simultaneous analysis of hundreds of different compounds in a label-free manner. In order to obtain a comprehensive metabolite and lipid data, a polarity switching MSI method using infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) was developed and optimized where the electrospray polarity was alternated from one voxel to the next. Healthy and cancerous ovarian hen tissue sections were analyzed using this method. Distribution and relative abundance of different metabolites and lipids within each tissue section were discerned, and differences between the two were revealed. Additionally, the utility of using mass spectrometry concepts such as spectral accuracy and sulfur counting for confident identification of analytes in an untargeted method are discussed.