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Abstract Duchenne Muscular Dystrophy (DMD) is a fatal genetic disorder currently having no cure. Here we report that culture substrates patterned with nanogrooves and functionalized with Matrigel (or laminin) present an engineered cell microenvironment to allow myotubes derived from non-diseased, less-affected DMD, and severely-affected DMD human induced pluripotent stem cells (hiPSCs) to exhibit prominent differences in alignment and orientation, providing a sensitive phenotypic biomarker to potentially facilitate DMD drug development and early diagnosis. We discovered that myotubes differentiated from myogenic progenitors derived from non-diseased hiPSCs align nearly perpendicular to nanogrooves, a phenomenon not reported previously. We further found that myotubes derived from hiPSCs of a dystrophin-null DMD patient orient randomly, and those from hiPSCs of a patient carrying partially functional dystrophin align approximately 14° off the alignment direction of non-diseased myotubes. Substrates engineered with micron-scale grooves and/or cell adhesion molecules only interacting with integrins all guide parallel myotube alignment to grooves and lose the ability to distinguish different cell types. Disruption of the interaction between the Dystrophin-Associated-Protein-Complex (DAPC) and laminin by heparin or anti-α-dystroglycan antibody IIH6 disenables myotubes to align perpendicular to nanogrooves, suggesting that this phenotype is controlled by the DAPC-mediated cytoskeleton-extracellular matrix linkage. Graphical abstract Image 1 Highlights • Non-diseased myotubes align perpendicular to nanogrooves on substrates having laminin. • Nanogrooved, laminin-modified substrates distinguish non-diseased and DMD myotubes. • This phenotypic biomarker may facilitate DMD drug development and early diagnosis. • All myotubes align parallel to grooves when grooves are larger or laminin is missing. • The DAPC and its interaction with laminin regulate perpendicular myotube alignment. [ABSTRACT FROM AUTHOR]