Introduction: β2-Adrenergic receptors (AR) are the primary subtype of adrenergic receptors found on blood leukocytes, and are recognized as key mediators of interaction between the sympathetic nervous system and immune system. This biological pathway is central in understanding neuroimmune signaling in a number of disease states, spanning mental disorders to traumatic brain injury. Traditionally, β2-ARs have been primarily assessed using radio-ligand binding assays, where results are limited in their ability to provide in depth cell-signaling information on specific leukocyte subtypes. As a result, current knowledge of β2-AR distribution, reactivity and potential implications in disease pathology or drug therapy are limited. This study evaluated β2-AR expression on circulating leukocyte subsets with various commercially available antibodies (Abs) using imaging cytometry. Methods: Whole blood was labeled with CD14 and CD16 surface markers for leukocyte subtype phenotyping. Four different β2-AR antibodies, sc-81578 – FITC (Santa Cruz), sc-81577 – PE (Santa Cruz), PA5-19649 (Pierce Biotechnology, USA) and MCA2784 (Serotec, USA) were assessed. DAPI stain was added to all samples to determine nuclear morphology. Based on cell area and aspect ratio, 100,000 single-cell events were acquired on an ImageStreamX Mark II® Cytometer (Amnis, Seattle, WA). Monocytes and lymphocytes were identified using side scatter (SSC) and CD14 expression. Neutrophils and eosinophils were identified using SSC and CD16. Results: β2−AR expression levels on leukocyte subpopulations in whole blood were detectable using image cytometery. Leukocyte subsets identified according to surface markers, size, granularity, cell and nuclear morphology displayed varying amounts of β2-AR expression. Furthermore, the PA5-19649 Ab performed best in terms of binding specificity and signal strength. Conclusion: Imaging cytometry is a valuable technique for evaluating β2-ARs on leukocyte subsets. The capability of this technology to provide in-depth, high throughput cell signaling information offers an attractive alternative to current methods used in the assessment of adrenergic receptors.