In recent biomedical research, images are generated at the high rates by using modern fluorescence microscopy. There are different image analysis software that can be used to perform a wide array of useful image analysis measurement. The identification of protein interaction is important for the detail study of localization and quantification of the expression. The main aim of the project was to evaluate the interactions of FUS/Plectin proteins in fibrosarcoma cells using the Blobfinder tool. The FUS/Plectin interaction localization was showed using In Situ Proximity ligation based assay (PLA) and these protein interactions were quantified by the Blobfinder tool. The obtained quantitative results from the tool were statistically analyzed by Graphpad prism software and Microsoft Excel. PLA results showed the localization of the FUS/Plectin interaction in the cytoplasm and on the border of nucleus. The Blobfinder tool obtained quantitative results which suggested that there were more interactions in the cytoplasm as compare to the nucleus. The histogram analysis also suggests that the frequency of interactions were higher in the cytoplasm when compared to nucleus in majority of cells. The obtained results from the Blobfinder tool and In situ PLA are in agreement with the results obtained from Immunoprecipitation pull down assay which shows FUS/Plectin interactions.