Small molecular inhibitors are excellent tools for manipulating cell reactions. They are widely used in scientific research to study molecular mechanisms of cells under physiological and pathophysiological conditions as well as in clinical applications to treat patients. However, their selectivity is often not well known. Moreover, it can vary according to cell types and the analysis methods used. Therefore, it is usually not possible to make comparisons between the data presented in the literature. Here we analyzed the selectivity of five chosen inhibitors of calcineurin/NFAT activation under the same conditions. Using a combination of fluorescent cell barcoding and phospho-specific flow cytometry we studied the inhibition of activation of NF-κBp65 and MAPK pathways in stimulated primary human Th cells. This semi-high throughput approach enabled us to demonstrate that (i) CsA and NCI3 are around 5 to 10- and 20-fold less potent, respectively, at inhibiting phosphorylation of NF-κBp65 and p38 than activation of NFAT, (ii) AM404 is at least 15-fold selective for NFAT but already toxic at concentrations above 40 μM, (iii) INCA6 is not selective at all, and (iv) BTP1 is at least 100-fold selective for inhibition of NFAT activation relative to NF-κBp65, p38 and ERK1/2 phosphorylation. Altogether, our results not only show the applicability of a semi-high throughput inhibitor test system but also that BTP1 is the most selective inhibitor of calcineurin/NFAT activation among the studied inhibitors under the used conditions.