Clinical laboratory testing for HER2 status in breast cancer tissues is critically important for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven analysis of tissue sections. We hypothesized that MALDI-IMS may determine HER2 status directly from breast cancer tissues. Breast cancer tissues (n = 48) predefined for HER2 status were subjected to MALDI-IMS, and protein profiles were obtained through direct analysis of tissue sections. Protein identification was performed by tissue microextraction and fractionation followed by top-down tandem mass spectrometry. A discovery and an independent validation set were used to predict HER2 status by applying proteomic classification algorithms. We found that specific protein/peptide expression changes strongly correlated with the HER2 overexpression. Among these, we identified m/z 8404 as cysteine-rich intestinal protein 1. The proteomic signature was able to accurately define HER2-positive from HER2-negative tissues, achieving high values for sensitivity of 83%, for specificity of 92%, and an overall accuracy of 89%. Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-abundance proteins.