We decellularized rat diaphragms with the main detergents 1% or 0.1% sodium dodecyl sulfate (SDS) and 4% sodium deoxycholate (SDC) by orbital shaking (OS) or retrograde perfusion (RP) through the vena cava. Our systematic comparison entails evaluation of decellularized diaphragmatic samples by (1) quantitative analysis including DNA quantification and biomechanical testing, (2) qualitative and semi-quantitative analysis by proteomics, as well as (3) qualitative assessment with macroscopic and microscopic evaluation by histological staining, immunohistochemistry and scanning electron microscopy.