Tumor necrosis factor superfamily cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) induces apoptosis in tumor cells by binding to death receptors DR4 and DR5 without affecting normal cells. However, the therapeutic use of TRAIL is limited, because many tumor cells are resistant to it. The resistance is partially related to interaction of TRAIL with the decoy receptors DcR1 and DcR2, which do not trigger the apoptotic signal and inhibit signaling of death receptors. Previously, we designed a unique DR5-specific TRAIL mutant variant DR5-B, which binds to DR5 receptor as effectively as the original cytokine, but has practically no interaction with DR4 and DcR1 receptors, and its affinity for DcR2 is reduced 400-fold. In the present work, the cytotoxity of TRAIL and DR5-B was analyzed on 12 different tumor cell lines and two types of normal cells. In nine of 12 tumor cell lines, DR5-B killed 1.5-5.0 times more tumor cells than TRAIL, and it did not exhibit toxicity towards normal cells. Chemotherapeutic drugs such as doxorubicin, paclitaxel, and bortezomib augmented the effect of both TRAIL variants, and the enhancing effect was more pronounced for DR5-B. Half-maximal effective concentrations (EC50) for DR5-B in combination with chemotherapeutic agents were 1.5-10.0 times lower than for wild-type TRAIL. Thus, DR5-B is a promising candidate both for monotherapy and in combination with chemotherapy for treatment of TRAIL-resistant tumors.