Use of flow cytometry to detect pluripotency markers on or in human embryonic stem cells (hESCs) is a powerful analytical tool. However, current staining methodologies for high-content analysis of large numbers of samples utilize large quantities of primary and secondary antibodies, are time consuming, and may suffer from sample-to-sample variability. To circumvent these issues, we have developed a reproducible, quick, and cost-effective method of staining 12 populations of hESCs grown under different conditions by labeling each with a unique optical signature (UOS). The UOS for each population is achieved by combining different combinations and concentrations of 3 esterase activated, live cell, fluorescent indicators. The individually stained populations are then combined and an aliquot of the hESC samples stained for pluripotency or other markers of interest in the far-red region of the spectrum. Based on the unique fluorescent intensity and emission wavelengths of each population, the characteristics of each population are decoded in software after flow cytometric analysis. We have validated both our staining procedure and decoding methods by mixing populations of differentiated and undifferentiated hESCs and successfully quantifying differences in the pluripotency markers SSEA-4, Tra-1-60, GCTM2, and CD9 between the 12 different populations. Our multiplexing approach allows for the addition of internal controls and reduces sample-to-sample variation, while offering a significant reduction in time and reagent consumption. We anticipate that this method will be of great benefit to laboratories conducting high-content flow cytometric analysis of hESCs.