The Proximity Ligation Assay (PLA) is a powerful method for visualizing protein–protein interactions with high specificity and sensitivity. By tethering DNA probes to antibodies directed against two target proteins, the PLA enables the detection of protein complexes within a 40-nm range. Upon colocalization of the target proteins, the DNA probes can anneal a connector oligonucleotide, initiating rolling circle amplification and generating a fluorescent signal detectable by microscopy. Here, we describe a detailed protocol for conducting a PLA in patient-derived tumor cells to visualize protein colocalization of two proteins in situ by fluorescence microscopy. With its ability to accurately capture protein–protein interactions and spatial organization within cells, the PLA is a valuable tool for insights into molecular mechanisms underlying brain tumor progression.