After the Chernobyl nuclear accident in 1986, numerous specimens of the so-called ‘fuel-containing masses’ or the Chernobyl ‘lava’ and hot particles were collected. Isotope analysis of Chernobyl specimens is the subject of special interest, since unexpected results on ²³⁸U/²³⁵U ratio has been reported previously. Although, over 30 years have passed since the Chernobyl accident, these samples are still a source of important information about the catastrophe as well as about the environmental behavior of highly radioactive materials. In the current study, glow discharge mass spectrometry with combined hollow cathode, pulse power supply and time-of-flight mass spectrometer was employed for the isotope analysis of uranium in Chernobyl-born microparticles. Six Chernobyl specimens (three crystals of artificial high-uranium zircon from the Chernobyl ‘lava’ and two hot particles) were analyzed. The method was optimized to cope the adverse oxide interferences. Simple isotope calibration with a single reference material of UO₂ with known ²³⁵U content (abundance, 1.80 ± 0.03%) was used. The validity of the method was checked using the sample with natural ²³⁵U abundance (0.72%) and by comparing with sector field inductively coupled plasma mass spectrometry. The relative error of ²³⁵U determination was ca. 1%, which is comparable to or excels the values, obtained by the competitive approaches (e.g. laser ablation inductively coupled plasma mass spectrometry, accelerator mass spectrometry or resonance ionization mass spectrometry). An important advantage of the developed method is the possibility to conduct direct analysis with partial preservation of varied solid materials without preliminary dissolution, separation or concentrating procedures. Other than partial sample preservation, this provides lower hazards to the analyst, owing to shorter sample handling time and lower probability of radioisotopes volatilization or their turning to aerosols.