Utrophin is an autosomal paralogue of dystrophin shown to functionally substitute for the loss of dystrophin in preclinical models of DMD. Ezutromid is a first-in-class small molecule that modulates the expression of utrophin in both DMD patient derived muscle cells and the dystrophin deficient mdx mouse model. Here we report the development and validation of cell and tissue based imaging approaches to help determine ezutromid’s mechanism of action and facilitate the identification of additional utrophin modulators. A high content cell based imaging approach has enabled the accurate measurement of utrophin protein in healthy and DMD patient derived muscle cell cultures. It also revealed a possible mechanism promoting differentiation and/or fusion in myoblasts treated with various utrophin modulators. In the mdx mouse, as with DMD patients, the regenerating skeletal muscle fibres express utrophin, making it difficult to distinguish drug derived utrophin modulation from utrophin expressed naturally during regeneration without employing additional pathology endpoints in parallel. We have developed an automated multiplexed immunohistochemical (IHC) assay with whole section digital tissue image analysis for the robust quantification of utrophin protein expression (intensity and % utrophin positive fibres). This approach distinguishes between utrophin expression due to drug modulation relative to that measured in control animal muscle. Combining the utrophin analysis with additional tissue morphometrics also provided a more informative measure of the effectiveness of utrophin modulating compounds. This is analogous to the muscle biopsy analysis performed by Flagship Bioscience in samples from Summit Therapeutics’ phase 2 clinical trial (PhaseOut DMD) evaluating ezutromid. Combining these preclinical approaches will assist us to better predict translational efficacy of utrophin modulation approaches in vitro and in the mdx mouse efficacy model.