To obtain extracellular matrix (ECM) of umbilical cord multipotent mesenchymal stromal cells (UC-MSCs), six decellularization protocols were used, based on physical (freeze/thaw) and chemical (Triton-X100, CHAPS, SDS, and NP-40) methods. The quality of decellularization was assessed using phase-contrast and fluorescence microscopy, quantitative image analysis was performed as well. It was shown that protocols based on CHAPS/NH4OH and Triton X-100/NH4OH are optimal for decellularizing UC-MSC layers and provide high-quality ECM. The SDS-based protocol may be useful when certain requirements to matrix structure are specified. The presented data will be in demand for the development of biomedical products.