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Abstract : A high-throughput clonogenic assay with a micropillar–microwell chip platform is proposed by using the colony area of glioblastoma multiforme (GBM) patient-derived cells (PDCs) from colony images. Unlike conventional cell lines, PDCs from the tumor are composed of heterogeneous cell populations, and some clonogenic populations form colonies during culture while the rest die off or remain unchanged, thus causing the diverse distribution of colony size. Therefore, area-based analysis of the total colonies is not sufficient to estimate total cell viability or toxicity responses. In this work, the average and standard deviation of an individual colony’s area calculated from the colony images were used as indicators for cell clonogenicity and heterogeneity, respectively. Two parameters (the total and average area of colonies) were compared to draw the colony’s growth curve and measure a doubling time and dose–response curve (IC50). Based on both analyses of two PDCs, 464T PDCs show a higher heterogeneity and clonogenicity than 448T PDCs. The differences in the doubling time and the IC50 according to the analysis methods suggest that the average area of colonies, rather than their total area, is suitable for heterogeneous and clonogenic samples.