Endoglin (CD105), a component of TGF-β, BMP-9, BMP-10 binding receptor complexes, is the marker of endothelial and mesenchymal stem cells. Its expression is significantly increased in the blood vessel endothelium of ischemic tissues and growing tumors. Measurement of the endoglin soluble form in serum and urine is used in diagnostics of cancer and pregnancy disorders. The aim of this work was to produce a panel of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. A recombinant protein derived from murine myeloma cells representing the whole extracellular part of endoglin was used as the immunogen. F1(SJL/J × BALB/c) mice were the donors of immune splenocytes. Hybridoma screening was performed using E. coli-produced recombinant molecules, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight different epitopes were produced. Eight antibodies bind the membrane-associated endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by Western blot. Sandwich ELISA system was designed to measure soluble endoglin in the cell culture medium.