Biocruces Health Research Centre 1 article published in JoVE Biochemistry Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software Rory Nolan1, Luis A. Alvarez1, Samuel C. Griffiths2, Jonathan Elegheert2, Christian Siebold2, Sergi Padilla-Parra1,2,3,4 1Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford, 2Division of Structural Biology, Wellcome Centre Human Genetics, University of Oxford, 3Dynamic Structural Virology Group, Biocruces Health Research Centre, 4IKERBASQUE, Basque Foundation for Science This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.