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<italic>Objective:</italic> Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for &#x2018;de novo&#x2019; regrowth or application as tissue &#x2018;patches&#x2019; that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat <italic>gastrocnemius medialis</italic> muscle through perfusion of the main feeding arteries. <italic>Results:</italic> Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) <italic>ex situ</italic> assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) <italic>in situ</italic> within the bioreactor environment assessment of the sample&#x2019;s passive mechanical elasticity. <italic>Conclusion:</italic> We find DNA and sarcomere-free constructs after 72 h of 0.1&#x0025; SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. <italic>Significance:</italic> Our <italic>MyoBio</italic> represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.

MyoBio: An Automated Bioreactor System Technology for Standardized Perfusion-Decellularization of Whole Skeletal Muscle