3D multicellular tumor spheroids (MCTSs) have recently emerged as a landmark for cancer research due to their inherent traits that are physiologically relevant to primary tumor microenvironments. A facile approach–laser‐ablated micro U‐wells–has been widely adopted in the past decade. However, the differentiation of microwell uniformities and the construction of arrays have all remained elusive. Herein, an improved laser‐ablated microwell array technique is proposed that can not only achieve arrayed MCTSs with identical sizes but can also perform high‐throughput drug assessments in situ. Three critical laser ablation parameters, including frequency, duty cycle, and pulse number, are investigated to generate microwells flexibly with a range from 170 to 400 μm. The choice of microwells is optimally arranged into an array via precise control of horizontal spacing (dx) and vertical spacing (dy) amenable of cell‐loss‐free culture during cell seeding. Harvested T24, A549 and Huh‐7 MCTSs from the microwell array correspond to approximately 75 to 140 μm in diameter. Anticancer drug screening of cisplatin validated IC50 values in 2D and MCTS conditions are 3.5 versus 9.1 μM (T24), 11.8 versus 277.7 μM (A549) and 33.5 versus 52.8 μM (Huh‐7), and the permeability is measured to range from 0.042 to 0.58 μm min−1.

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