Background: Vascular Endothelial Growth Factor A (VEGF-A) is a key mediator of angiogenesis, primarily signalling via VEGF Receptor 2 (VEGFR2). Endothelial cells also express the co-receptor Neuropilin-1 (NRP1) that potentiates VEGF-A/VEGFR2 signalling. VEGFR2 and NRP1 had distinct real-time ligand binding kinetics when monitored using Bioluminescence Resonance Energy Transfer (BRET). We previously characterised fluorescent VEGF-A isoforms tagged at a single site with tetramethylrhodamine (TMR). Here, we explore differences between VEGF-A isoforms in living cells that co-expressed both receptors. Experimental Approach: Receptor localisation was monitored in HEK293T cells expressing both VEGFR2 and NRP1 using a membrane-impermeant HaloTag and SnapTag technologies. To isolate ligand binding pharmacology at a defined VEGFR2/NRP1 complex, we developed an assay using NanoBiT complementation technology whereby heteromerization is required for luminescence emissions. Binding affinities and kinetics of VEGFR2-selective VEGF165b-TMR and non-selective VEGF165a-TMR were monitored using BRET from this defined complex. Key Results: Cell surface VEGFR2 and NRP1 were co-localised and formed a constitutive heteromeric complex. Despite being selective for VEGFR2, VEGF165b-TMR had a distinct kinetic ligand binding profile at the complex that largely remained elevated in cells over 90 minutes. VEGF165a-TMR bound to the VEGFR2/NRP1 complex with kinetics comparable to those of VEGFR2 alone. Using a binding-dead mutant of NRP1 had no impact on the binding kinetics or affinity of VEGF165a-TMR. Conclusions and Implications: This NanoBiT approach enabled real-time ligand binding to be quantified in living cells at 37°C from a specified complex between a receptor tyrosine kinase and its co-receptor for the first time.