In this report, we present the implementation of mass cytometry for intracellular staining using fixed whole blood. In our assay described here, 250 µl of whole blood, is stimulated in vitro with PMA/ionomycin (or left unstimulated), in the presence of secretion inhibitors (brefeldin A and monensin), lysed-fixed using SMART TUBE buffers, barcoded (optional), surface stained, fixed, stained for intracellular markers, fixed and DNA stained. Using 250 µl of whole blood from a healthy donor, we show that the expression of major lineage populations such as T cells, B cells, NK cells and monocytes, as well as cytokines such as CD4⁺ and CD8⁺ IFNγ and TNFα across multiple batches (n = 27) is consistent, with the co-efficient of variation (CVs) ≤ 21%, implying minimum inter-variability. For each major cell type, the percentage is reported as a percent of singlets. The percentage of cytokine expression in response to stimulation is reported as a percent of the immediate parent cell type. This protocol has a number of benefits: from a biological perspective, it can be applied to clinical studies especially where blood draw volumes are limiting. Technically, the protocol can be adapted for barcoding, which adds the benefits of more uniform sample staining as well as antibody conservation especially for large study cohorts. Finally, for studies involving infectious diseases including the current global COVID-19 pandemic, this protocol permits infectious samples to be fixed prior to processing and staining, thereby reducing biosafety risks.