Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising new anticancer biotherapeutic. As shown by many preclinical studies, TRAIL efficiently induces apoptosis in numerous tumor cell lines but not in the majority of normal cells. However, an increasing number of publications report on a predominance of TRAIL resistance in primary human tumor cells, which require sensitization for TRAIL-induced apoptosis. Sensitization of cancer cells by treatment with chemotherapeutic drugs and irradiation has been shown to restore TRAIL sensitivity in many TRAIL-resistant tumor cells. Accordingly TRAIL treatment has been successfully used in different in vivo models for the treatment of tumors also in combination with chemotherapeutics without significant toxicity. However, some reports demonstrated toxicity of TRAIL alone or in combination with chemotherapeutic drugs in normal cells. This review summarizes data concerning the apoptosis-inducing pathways and efficacy of TRAIL, alone or in combination with chemotherapeutic drugs, in primary cancer cells compared to the unwanted effects of TRAIL treatment on normal tissue. We discuss the different in vitro tumor cell models and the potential of different recombinant forms of TRAIL or agonistic antibodies to TRAIL death receptors. Most preclinical studies show a high efficiency of a combinatorial TRAIL-based therapy in animal models and in primary human ex vivo tumor cells with a low toxicity in normal cells. Accordingly clinical phase I/II studies have begun and will be developed further with caution.