To develop better vaccines and more targeted treatments for cancer and autoimmune disorders, the disease-specific T cells and their cognate antigens need to be better characterized. For more than two decades, peptide-major histocompatibility complex (pMHC) tetramers and flow cytometry have been the gold standard for detection of CD8+ and CD4+ T cells specific to antigens in the context of MHC class I and class II, respectively. Nonetheless, more recent studies combining such reagents with mass cytometry, i.e. cytometry by time of flight (CyTOF), have offered far more comprehensive profiling of antigen-specific T-cell responses. In addition, mass cytometry has enabled ex vivo screening of CD8+ T-cell reactivities against hundreds of MHC class I restricted candidate epitopes. MHC-class II molecules, on the other hand, have been challenging to combine with mass cytometry as they are more complex and bind with lower affinities to cognate T-cell receptors than MHC-class I molecules. In this review, I discuss how techniques originally developed to improve the staining capacity of pMHC tetramers in flow cytometry led to the successful combination of such reagents with mass cytometry. Especially, I will highlight very recent advances facilitating the combination with pMHC-class II tetramers. Together, these mass cytometry-based studies can help develop more targeted treatments for cancer and autoimmune disorders. This article is protected by copyright. All rights reserved.