The advent of mass cytometry has facilitated highly multi‐parametric single‐cell analysis allowing for the deep assessment of cellular diversity. While the data and analytical power of this approach are well described, associated technical and experimental hurdles remain. Issues like equipment breakdown and sampling of large‐scale batches, which may require multiple days of data acquisition, are minor but critical obstacles that prompt a technical solution, especially when dealing with precious samples. An ability to cryopreserve mass cytometry samples that have already been stained would alleviate numerous technical limitations we face with currently used sample‐handling approaches. Here, we evaluated two protocols for freezing of already‐stained and fixed cellular samples and compared them with standard sample refrigeration in staining buffer. A comprehensive human T cell staining phenotypic and functional profiling panel was used and the signal intensity and reliability of each marker was assessed over a 4‐week period. In general, cellular viability, DNA Ir‐Intercalator and barcode staining were minimally affected by freezing compared to refrigeration, and the signal intensities for cell surface markers and receptors were not compromised. Intracellular cytokine staining did show some decreases in signal intensity after freezing, with the decreases more prominent in a methanol‐based protocol compared to a protocol involving the use of 10% DMSO in FBS. We conclude that freezing already‐stained samples suspended in 10% DMSO in FBS is practical and efficient way to preserve already‐stained samples when needed. © 2016 International Society for Advancement of Cytometry.