Improving our knowledge of the precise mechanisms by which cells signal is vital for advancing our overall understanding of cellular physiology and for the development of viable new therapeutics. The recent identification of endosomal-propagated signalling in select G protein-coupled receptors (the “signalling endosome hypothesis”) has broadened our view of the complexity of receptor signalling mechanisms. However, our understanding of the precise mechanisms by which endosomal signalling occurs, as well as its functional significance and potential for therapeutic exploitation is currently very limited. Here we describe the synthesis and testing of a new probe to investigate neurokinin-1 receptor (NK1R) signalling from endosomes. The probe, synthesised via solid-phase techniques, incorporates a peptidic NK1 receptor antagonist (spantide), and endosomal uptake-promoting group (cholestanol), and a fluorescent dye for visualisation (sulfo-Cy5). Initial testing has confirmed that the probe accumulates within endosomes of HEK293 cells, with further testing underway to investigate what effects arise from antagonism of NK1R within these endosomes.