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Background Hyperthermia is one of the new and still poorly known methods used in cancer treatment. It consists of raising the patient's body temperature for therapeutic purposes. The article presents the results of in vitro studies describing the effect of an elevated temperature of 39.5°C, the busulfan cytostatic and their combination on the level of apoptosis of human leukemia HL-60 cells. Objectives During the experiments, the influence of a 2.5°C temperature increase on the behavior of the population of 2 groups of HL-60 cells, with busulfan cytostatic and without the cytostatic, was investigated. The control group consisted of 2 groups of HL-60 cells incubated at 37.0°C with the cytostatic and without the cytostatic. Two questions were asked: 1. Is low-temperature hyperthermia likely to have an effect on the effectiveness of busulfan cytostatic? 2. Does the increase in temperature by 2.5°C have an effect on the level of apoptosis in the unsaturated HL-60 cell line? Material and methods Human promyelocytic leukemia cell line HL-60 was used in the experiments to examine the influence of temperature on apoptosis HL-60 in 2 separated incubators set to 37.0°C and 39.5°C for 3 h. Apoptosis was assessed with flow cytometry using Annexin V. Results An increase in mortality of HL-60 cells was found in the case of simultaneous exposure to elevated temperature and busulfan in comparison to the group of cells treated with the cytostatic alone. There was no observed effect of an elevated temperature of 39.5°C alone on the level of HL-60 cell apoptosis. Conclusions Analysis of the study results indicates that low-temperature hyperthermia may be used to increase the effectiveness of busulfan treatment. No effect of an elevated temperature of 39.5°C on the level of apoptosis in HL-60 cells that were not treated with busulfan was observed. There is a need to test the efficacy of other cytostatic agents at elevated temperatures.