Histamine and its release can be studied by multicolor flow cytometry on a single cell level by an enzyme affinity method (HistaFlow®). However, for the time-being, the clinical and scientific application of the HistaFlow® technique remains limited. This study aims at verifying the reliability of the HistaFlow® as an instrument to quantify IgE-mediated basophil responses to drugs, i.e., rocuronium, which are believed to be less potent basophil activators than large proteinaceous allergens. Ten patients and three exposed control individuals were included in this study. Each subject underwent in vitro basophil activation tests (HistaFlow®) with 0.16 and 1.6 mmol/L rocuronium. Patients showed an activation of basophils ranging from 11 to 86% of CD63 positive basophils and a median histamine release per cell from 68 to 100% after stimulation with an optimal concentration of 1.6 mmol/L rocuronium. For the control individuals no activation was demonstrable. This study confirms that the HistaFlow® technique is a reliable tool to study histamine release by individual cells in response to drugs. Although the HistaFlow® technique will probably not add to the diagnostic management of rocuronium allergy, our findings suggest that the technique could constitute an important asset for future studies on the pathomechanism(s) of immediate drug hypersensitivity reactions. © 2015 International Clinical Cytometry Society.