Time-domain diffuse reflectance spectroscopy (TRS), a medical sensing technique, was used to evaluate internal kiwi fruit quality. The application of this pulsed laser spectroscopic technique was studied as a new, possible non-destructive, method to detect optically different quality parameters: firmness, sugar content, and acidity. The main difference with other spectroscopic techniques is that TRS estimates separately and at the same time absorbed light and scattering inside the sample, at each wavelength, allowing simultaneous estimations of firmness and chemical contents. Standard tests (flesh puncture, compression with ball, .Brix, total acidity, skin color) have been used as references to build estimative models, using a multivariate statistical approach. Classification functions of the fruits into three groups achieved a performance of 75% correctly classified fruits for firmness, 60% for sugar content, and 97% for acidity. Results demonstrate good potential for this technique to be used in the development of new sensors for non-destructive quality assessment.