The ILF (Image Landscapes’ Fractal Dimension) method and [Formula: see text] method obtained by a 2D generalization of Higuchi’s algorithm were applied to a set of 120 digital histological images of Anal Intraepithelial Neoplasia (AIN). The main goal of this research was to examine accuracy that means sensitivity and specificity of these methods and compare the applicability of both methods in the quantitative characterization and differentiation of clinical cases of AIN. Histological examination by an experienced pathologist revealed three grades of AIN tumors in the 120 histological slices: 36 of AIN1, 56 of AIN2 and 28 of AIN3. Statistical tests showed significant differences between calculated fractal dimension values in three datasets (AIN1, AIN2 and AIN3) using ILF and [Formula: see text] methods at the level of significance of 0.05. Application of the ILF and [Formula: see text] methods has an advantage when it comes to speed, accuracy, simplicity and time necessary for analysis. Both methods can be successfully applied for differentiation between AIN stages giving practically the same results. They can easily be adapted to other histological specimen.