Neuropeptide Y1 Receptor (NPY1R), a G protein coupled receptor, is essential for regulating appetite, energy balance, emotional responses and is implicated in pathological disorders like obesity, anxiety and cancers. Currently, only small molecule reagents are available for assessing the expression of NPY1R in cancers [1]. This study provides evidence in support for validation of two antibodies that bind separate epitopes on the extracellular domain of human NPY1R with the potential for improved imaging and/or treatment, focusing first on breast cancers [2]. The estrogen receptor (ER) exerts a positive effect on the activity of the NPY1R either at mRNA expression or protein activation. The immediate aim was the validation of two monoclonal antibodies, mAb2A10 and mAb8D6, with a COS-7 cell line, developed to over-express human NPY1R, and compared to MCF-7 cells derived from breast cancer. Secondly, stripped estrogenic media, which influences the potential role of ER in activation of NPY1R, was also investigated. The FLAG-NPY1R construct was introduced into COS-7 cells via plasmid transfection, followed by successive selection with puromycin to establish a stable cell line, designated as 10B.Using flow cytometry, an anti-FLAG:568 antibody was used to identify a high NPY1R expressing clone, D9, which was used for experiments. Western blotting using either antibody identified an ~136 kDa band (homodimer) in both the high NPY1R-expressing D9 clone and MCF-7 cells. This was absent when cells were grown in non-estrogenic media (phenol red free, charcoal stripped FBS), indicating that the estrogenic effect controls activation of the NPY1R protein rather than expression of mRNA. Confocal microscopy images supported these results, confirming the antibodies' specificity and receptor localization in the D9 clone. Therefore, the antibody validation process confirmed the specificity of both mAb2A10 and mAb8D6 for the FLAG-NPY1R. These findings collectively validate the antibodies' potential as diagnostic or therapeutic tools for identifying NPY1R-expressing cancers.