Leukemia is a very common cancer worldwide, and different drugs have been applied to treat the disease. However, the influence of the drugs on the biomechanical properties of leukemia cells, which are related to the risk of leukostasis, is still unknown. Moreover, accurate measurement of biomechanical properties of leukemia cells is still a challenging task because of their non-adherent nature and high sensitivity to the surrounding physiological conditions. In this study, a protocol to measure the biomechanical properties of leukemia cells by performing indentation tests using optical tweezers is proposed. The biomechanical properties of normal leukemia cells and cells treated with various cancer drugs, including phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRa), Cytoxan (CTX), and Dexamethasone (DEX), were measured. The adhesion between the cells and certain proteins existing in the extracellular matrix, i.e., fibronetin and collagen I, was also characterized with the help of a static adhesion assay. It was found that after treatment by ATRa, CTX, and DEX, the cells became softer, and the adhesion between the cells and the proteins became weaker. PMA treatment caused no change in the stiffness of the HL60 cells, but increased the stiffness of the K562 cells, and increased the cell–protein adhesion of both K562 cells and HL60 cells.