Single-cell, spatially resolved omics analysis of tissues is poised to transform biomedical research and clinical practice. We have developed an open-source, computational histology topography cytometry analysis toolbox (histoCAT) to enable interactive, quantitative, and comprehensive exploration of individual cell phenotypes, cell-cell interactions, microenvironments, and morphological structures within intact tissues. We highlight the unique abilities of histoCAT through analysis of highly multiplexed mass cytometry images of human breast cancer tissues.