Measurement of virus concentration is essential for effective virus-based transfection technologies. Here, we describe a user-friendly, image-based cell-size estimation (ICSE) assay for baculovirus quantification that relies on automated determination of cell diameters from bright-field microscopy images. In the ICSE assay, microplate-based imaging systems and our custom ICSE-Tools software enable measurement of cell morphological parameters over time. Results from the ICSE assay were in agreement with virus concentration measurements obtained using the traditional plaque assay as well as the Coulter principle-based cell-size measurement assay. ICSE-Tools is designed for data organization and image analysis from microplate-based imaging systems, and is freely available at www.gpcr.ut.ee/software.html.