Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of 'kiss-and-run' interactions between immune cells 'Labelling Immune Partnerships by SorTagging Intercellular Contacts' (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4+ T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.