QBEND/10 is a mouse immunoglobulin lambda-chain monoclonal antibody with strict specificity against human hematopoietic progenitor cell antigen CD34. Our in vitro study showed that QBEND/10 impairs the tube formation of human umbilical vein endothelial cells (HUVECs)， suggesting that the antibody may be of potential benefit in blocking tumor angiogenesis. We provided a de novo protein sequencing method through tandem mass spectrometry to identify the amino acid sequences in the variable heavy and light chains of QBEND/10. To reduce immunogenicity for clinical applications， QBEND/10 was further humanized using the resurfacing approach. We demonstrate that the de novo sequenced and humanized QBEND/10 retains the biological functions of the parental mouse counterpart， including the binding kinetics to CD34 and blockage of the tube formation of the HUVECs.