New generations of treatments successfully target cancer cell signaling and modulate anti-cancer immunity to improve outcomes for advanced-stage malignancies. Such clinical responses are governed by an evolving milieu of cancer, immune, and stromal cell subpopulations. Mass cytometry is particularly well-suited to track cells in complex tissues because 35-plus measurements can be made on each of hundreds of thousands of cells per sample. This talk will focus on mass cytometry as an example of systems-level characterization of cells in healthy human tissues and solid tumors. The state of the art in single-cell cancer biology will be discussed, including tissue collection, technical and biological quality controls, analysis of cell signaling (phospho-flow) and computational analysis. Mass cytometry holds great promise for dissecting cellular microenvironments, monitoring treatment impact, revealing cellular biomarkers and effector mechanisms and creating new treatments that productively engage the immune system.