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Hyperthermia affects certain regulatory proteins, kinases or cyclins, resulting in alternations to the cell cycle and even to apoptosis. Damage to the cell plasma membrane is a key factor in the killing of a cell by hyperthermia. Analysis at the single-cell level is necessary for understanding the fundamental mechanisms of hyperthermia-induced cell death and the generation of thermotolerance in surviving cells. Engineering approaches achieving precise control of cellular micropatterning provide the potential for investigating the mechanisms of thermal injury to cells at the single-cell level. The main purpose of this study is to fabricate a hydrogel chip with microwells for cellular patterning and to demonstrate the feasibility of measurement of supraphysiological thermal injury in human carcinoma cells (HeLa cells) at the single-cell level. To accomplish this, measurement of membrane injury by dye leakage post-thermal insult was performed and reported in this work. A hydrogel chip with microwells with different diameters was fabricated. For cell concentrations at 0.5 × 106 cells/mL, the occupancy of cells on the microchip with 40 μm microwells was up to 86.6%, a value far higher than that found on the 30 μm microwells (approximately 78.5%). Most microwells of 30 μm in diameter (about 70%) were occupied by a single cell. The fluorescent images showed that calcein leakage occurred when cell membranes were damaged under supraphysiological temperatures between 43 and 50°C. The normalized intensity of calcein decreased to 32% under a supraphysiological temperature of 43°C for 20 min. The intensity of calcein in cells was less than 20% under a supraphysiological temperature of 50°C. The feasibility of the single-cell-based experiment of thermal injury in the microchip with hydrogel microwells was therefore successfully demonstrated.