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Based on four thawing methods (still air, still water, ultrasonic wave, and microwave) and single-factor tests, we established a four-factor three-level response surface methodology for a regression model (four factors: pH, drip loss rate, cooking loss rate, protein content). The optimal combined thawing method for beef rib-eye is: microwave thawing (35 s work/10 s stop, totally 170 s) until beef surfaces soften, then air thawing at 15°C until the beef centre temperature reaches -8°C, and finally ultrasonic thawing at 220 W until the beef centre temperature rises to 0°C. With this method, the drip loss rate is 1.9003%, cooking loss rate is 33.3997%, and protein content is 229.603 μg, which are not significantly different from the model-predicted theoretical results (P ≥ 0.05).