Facioscapulohumeral dystrophy (FSHD) is caused by the loss of repression at the D4Z4 locus on chromosome 4. This leads to an altered gene expression program in skeletal muscle and muscle fiber cell death caused by aberrant expression of the homeobox transcription factor DUX4. Using novel in vitro culture systems and highly sensitive assays to detect DUX4 and its downstream genes, we screened an annotated chemical library of >1000 biologically active compounds and identified a p38α/β small molecule inhibitor, losmapimod, as a novel regulator of DUX4 expression in FSHD myotubes. Using optimized myotube culture conditions, we observed that losmapimod treatment results in reduction of DUX4 expression, activity, and cell death in FSHD patient-derived myotubes. Due to the p38α/β pathway having been linked to muscle cell differentiation, we further examined the effect of losmapimod treatment on myotube formation. We developed a quantitative assay to assess skeletal muscle differentiation in vitro, which showed losmapimod did not impact myogenesis. We also further assessed gene expression changes in FSHD myotubes upon p38α/β inhibition. RNA-sequencing analyses showed a DUX4 program-specific rescue following losmapimod administration to FSHD myotubes. It was observed that only a small number of genes were differentially expressed after treatment with the majority being targets of DUX4, with no negative impact on key drivers of myogenic programing. These in vitro findings highlight the potential of Losmapimod for the treatment of FSHD, a condition that today has no approved therapies.