The current study investigates the use of a non-destructive hyperspectral imaging approach for the evaluation of kiwifruit cv. “Hayward” internal quality, focusing on physiological traits such as soluble solid concentration (SSC), dry matter (DM), firmness, and tannins, widely used as quality attributes. Regression models, including partial least squares regression (PLSR), bagged trees (BTs), and three-layered neural network (TLNN), were employed for the estimation of the above-mentioned quality attributes. Experimental procedures involving the Specim IQ hyperspectral camera utilization and software were followed for data acquisition and analysis. The effectiveness of PLSR, bagged trees, and TLNN in predicting the firmness, SSC, DM, and tannins of kiwifruit was assessed via statistical metrics, including R squared (R²) values and the root mean square error (RMSE). The obtained results indicate varying degrees of efficiency for each model in predicting kiwifruit quality parameters. The study concludes that machine learning algorithms, especially neural networks, offer substantial accuracy, surpassing traditional methods for evaluating kiwifruit quality traits. Overall, the current study highlights the potential of such non-destructive techniques in revolutionizing quality assessment during postharvest by yielding rapid and reliable predictions regarding the critical quality attributes of fruits.