Dystrophin restoration through microdystrophin gene addition therapy is a major ongoing clinical trial approach for treating DMD. However, to date, major milestones have not been met raising concerns of efficacy. One possibility underlying the lack of efficacious clinical translation centers on uDys protein stability in vivo. We generated a gene regulated system of transgenic animal models expressing μDys (ΔR4–R23, ΔCT) constructs in heart and skeletal muscle, respectively, to investigate μDys post-translational modifications (PTMs), and to identify pathways that could promote μDys stabilization in vivo. Data show markedly reduced half-life of μDys in skeletal and heart muscles (~5 days) as compared to parallel studies of full-length dystrophin showing long half-life (~6 months) in heart and skeletal muscles in vivo. Pharmacologic inhibition of the proteasome system using bortezomib significantly increased μDys levels in muscle in vivo, indicating the ubiquitin-proteasome system as a key determinant of μDys turnover in muscle in vivo. Data show that μDys protein instability in vivo is accompanied by accumulation of Dys immunopositive high molecular weight bands indicative of PTMs. We used mass spectrometry to characterize μDys PTMs under in vivo conditions. These analyses identified multiple phosphorylation and ubiquitination sites distributed along the construct, with modification clusters localized within the R3 domain and the cysteine rich region of the truncated dystrophin protein that differed from in silico/ curated database data of full-length dystrophin in vivo. These findings were independently validated by Pro-Q Diamond phosphoprotein staining and immunoprecipitation assays, collectively indicating that μDys undergoes extensive and region-specific post-translational regulation that could in turn affect stability and function. In parallel studies, E3 ligases MuRF-1 and Atrogin-1 were upregulated in μDys transgenic skeletal muscles compared to full-length dystrophin, supporting the enhanced ubiquitin mediated turnover. In summary, this work provides the first data set of μDys PTMs’ in vivo, establishing that truncated dystrophin instability is regulated through UPS-dependent degradation in vivo. By uncovering the molecular determinants of μDys protein instability in vivo, these findings establish the investigational framework for increasing microdystrophin protein content levels by extending protein half-life in dystrophic muscle in vivo.