Calcium flux measurement is a crucial assay in lymphocyte activation. However, with the currently well established flow cytometric methods, it is a tedious procedure that is difficult to control to avoid variation between samples. This leads to unwanted sources of error that can make it problematic to interpret the results. Here we present an improved method that allows different cell populations to be tested in the same sample. Samples are pre-labeled with CFSE or Cy5 then mixed and stimulated to induce calcium flux. This facilitates more rapid and accurate measurement of calcium flux and also dramatically reduces the cost and effort required for this type of assay.