Analysis of large biomolecules including proteins has proven challenging using ambient ionization mass spectrometry imaging techniques. Here, we have successfully optimized desorption electrospray ionization mass spectrometry (DESI-MS) to detect intact proteins directly from tissue sections and further integrated DESI-MS to a high field asymmetric waveform ion mobility (FAIMS) device for protein imaging. Optimized DESI-FAIMS-MS parameters were used to image mouse kidney, mouse brain, and human ovarian and breast tissue samples, allowing detection of 11, 16, 14, and 16 proteoforms, respectively. Identification of protein species detected by DESI-MS was performed on-tissue by top-down ultraviolet photodissociation (UVPD) and collision induced dissociation (CID) as well as using tissue extracts by bottom-up CID and top-down UVPD. Our results demonstrate that DESI-MS imaging is suitable for the analysis of the distribution of proteins within biological tissue sections.