The purpose of the current study was to develop an optimized PLA protocol for non-adherent cells using commercially available materials and kits, and common molecular and cellular biology laboratory materials. By taking the example of ETV6-RUNX1 fusion protein, we present an improved method for the detection of fusion proteins and their partners that can be an important tool in scientific and translational research. PLA is an innovative method of protein-protein colocalization detection by molecular biology that combines the advantages of microscopy, specifically the requirement of few cells and in situ visualization, with the advantages of molecular biology precision, enabling detection of protein proximity theoretically ranging from 0 to 40 nm. We provide compelling negative and positive controls, and demonstrate that the optimized PLA procedure is sensitive to total protein level.