For the past few years, research has focused on the application of Near Infrared Spectroscopy (NIR) techniques to analyze the quality of apple varieties. As all fruit quality and nutritional parameters are affected by many factors, to standardize the sampling methods prior to analysis is very important. In this study, the intra-fruit variability of apple varieties in terms of quality parameters (sugar, total polyphenol and vitamin C content) was examined. The adequacy of the protocols usually applied to analyze apple quality (reference analyses of a representative sample of the apple and NIR measurements collected at four points 45° from each other in the equatorial region of the apple) was then investigated. The reference values for the quality parameters showed important intra-fruit variability for all quality parameters analyzed. The results also showed that there was little difference between the mean value at the four points and the mean value of the entire apple. This suggested that a mean value obtained from a representative sample of an apple is enough to assess the variability within the fruit and to estimate, with precision, the content of the quality parameters. The results of NIR spectroscopy showed that measurement at the four target points is suitable for predicting quality parameters precisely. The study concluded that standardized reference analyses should be done on a representative fruit sample of an apple and spectral measurements made at four points 45° from each other in the equatorial region of the apple.