Tendons and ligaments play an important role to ensure mobility and stability. To correctly understand the characteristics of these fibrous collagenous connective tissues, it is fundamental to highlight their 3D microstructure. In this study a microtomography (microCT) system was used to acquire human hamstring tendons after performing specific preparations to enhance image contrast. Specifically, samples were treated either through chemical dehydration or by 2% of phosphotungstic acid ([Formula: see text]) in water ([Formula: see text]) or in 70% ethanol ([Formula: see text]) solution. Acquired images were elaborated using dedicated techniques based on 3D Hessian multiscale filter so as to highlight the fibrous structure and identify specific geometric features. For any strategy of sample preparation, the proposed approach resulted to be adequate for identifying fascicle features, thus obtaining structures with diameter in the range of 100–600 [Formula: see text] and proper longitudinal alignment. In conclusion, a novel contrast enhancement microCT protocol was designed and preliminarily validated for the microstructural analysis of fibrous tissues.