Lymphedema is a pathology caused by poor lymphatic flow which may lead to complete disability. Currently, precise, non-invasive techniques for quantifying lymphedema are lacking. In this paper, the results of an in vivo assessment of lymphedema via a developed small-animal model using the hindlimbs of rats and an optical coherence tomography (OCT) technique are presented. This model of lymphedema was based on a surgical lymph node resection and subsequent two-step X-ray exposure. The development of lymphedema was verified via the histological examination of tissue biopsies. The properties of the lymphedematous skin were analyzed in vivo and compared with healthy skin via OCT. The main differences observed were (1) a thickening of the stratum corneum layer, (2) a thinning of the viable epidermis layer, and (3) higher signal attenuation in the dermis layer of the lymphedematous skin. Based on the distribution of the OCT signal’s intensity in the skin, a machine learning algorithm was developed which allowed for a classification of normal and lymphedematous tissue sites with an accuracy of 90%. The obtained results pave the way for in vivo control over the development of lymphedema.