Mass cytometry, or Cytometry by Time-Of-Flight (CyTOF), is a powerful platform for high-dimensional single-cell analysis of the immune system. It enables the simultaneous measurement of over 40 markers on individual cells through the use of monoclonal antibodies conjugated to rare-earth heavy metal isotopes. Coupled with our already extensive immunological knowledge of canonical immune subsets and an ability to delve into and describe subtle populations, mass cytometry presents an opportunity to investigate cumulative subtle changes across many specific immune subsets in a range of clinical cohorts. Primarily we look at peripheral blood mononuclear cells, to assess systemic perturbations evident to reveal disease specific immune signatures, with the benefit that this analysis platform strategy facilitates cross cohort analysis. Aspects of the mass cytometry workflow including antibody panel development, sample preparation, technical challenges and effective multi-parametric data analysis strategies as applied to varied clinical cohorts will be discussed.