We aimed to establish a fluorescence intensity-based colony area sweeping method by selecting the area of highest viability among patient-derived cancer cells (PDC) which has high tumor heterogeneity. Five gastric cancer cell lines and PDCs were screened with 24 small molecule compounds using a 3D micropillar/microwell chip. 100 tumor cells per well were immobilized in alginate, treated with the compounds, and then stained and scanned for viable cells. Dose response curves and IC50 values were obtained based on total or selected area intensity based on fluorescence. Unlike homogeneous cell lines, PDC comprised of debris and low-viability cells, which resulted in an inaccurate estimation of cell viability using total fluorescence intensity as determined by high IC50 values. However, the IC50 of these cells was lower and accurate when calculated based on the selected-colony-area method that eliminated the intensity offset associated with the heterogeneous nature of PDC. The selected-colony-area method was optimized to accurately predict drug response in micropillar environment using heterogeneous nature of PDCs.