The molecular characterisation of complex protein-protein interactions is vital for understanding cellular signalling pathways and improving targeted drug development. Toll-like receptor 4 (TLR4) is the innate immune receptor for bacterial lipopolysaccharide (LPS) and is implicated in bacterial sepsis and many inflammatory diseases. Understanding the interactions that specifically mediate TLR4 signalling pathways can aid in the development of new anti-inflammatory drugs. Upon TLR4 activation at the cell membrane, adaptor proteins MAL and MyD88 are recruited through toll-interleukin receptor (TIR) domain interactions to form a signalosome, leading to the activation of transcription factors including NF-κB. We provide insights into TIR domain interactions by structural studies of large filamentous assemblies that spontaneously form in vitro. Establishing the biological relevance of the interaction interfaces required analysis of the signalling function of interface mutant proteins. We developed a flow cytometry-based fluorescence assay for rapid analysis of TLR4 or MyD88 mutants. HEK293-NF-κB driven mScarlet-I fluorescent reporter cell lines, lacking the target protein but containing a complete signalling pathway, were generated. Transient transfection of wild-type and mutant TLR4-GFP or GFP-MyD88 constructs allowed detection of both protein expression level and signalling activity in individual cells by flow cytometry. This evades the problems of variable transfection efficiency as analysis can be restricted to cells with appropriate levels of TLR4/MyD88 expression. Furthermore, predictions of structural instability and possible mutation-related misfolding can be made. The robust signalling data we generated supports the interactions predicted by in vitro structures, with an exclusive head-to-tail dimerisation of TLR4 TIR domains and lateral recruitment of MAL. In addition, the importance of the intrastrand interaction interface in the MyD88 TIR domain filament for functional signalling is demonstrated. Furthermore, the assay can be adapted to suit a multitude of signalling pathways and provide substantial improvements on commonly used methods for structure-function analysis.