Patient-derived organoids (PDOs) are valuable for predicting the anticancer efficacy of therapeutics. However, conventional Matrigel-based PDO culture systems impede cellular therapy testing by preventing direct contact between cell therapies and organoids. In this study, we present a novel microwell array for culturing Matrigel-free PDOs to evaluate the in vitro anticancer activity of chimeric antigen receptor T (CAR-T) cells. We fabricated the microwells with a multi-layer nested architecture using a molding process, in which the casting molds were precision-customized via rapid three-dimensional (3D) printing. This microwell system enables the cultivation of organoids without Matrigel. Using this platform, we successfully cultured Matrigel-free lung cancer organoids (LCOs) derived from lung adenocarcinoma patients harboring epidermal growth factor receptor (EGFR) mutations. Similar to primary cancer cells, the cultured LCOs exhibited high EGFR protein expression. EGFR-CAR-T cells efficiently targeted and lysed Matrigel-free LCOs in the microwells but failed to kill conventionally cultured LCOs grown in Matrigel. Furthermore, EGFR-CAR-T cells effectively inhibited the growth of POD xenograft (PDOX) tumors in vivo, consistent with the in vitro LCO results. These findings demonstrate that microwell-cultured, Matrigel-free LCOs provide an in vitro platform for assessing CAR-T cell efficacy, paving the way for standardized organoid models in cell therapy development.