Analytical method for the determination of ultra-trace levels of nitro-PAHs in various biotic matrices has been developed. Soxhlet extraction and/or solvent extraction enhanced by sonication were used for isolation of target analytes; GPC followed by SPE were employed for purification of crude extracts. GC-MS/NCI technique was utilised for identification/quantitation of target analytes. Performance characteristics of implemented method were obtained through thorough in-house validation procedure. The main sources of uncertainties were critically evaluated, possible strategies of their elimination/minimisation were considered and consequently employed. Examination of real-life samples of various foodstuffs (complete human diet, mate tea, pumpkin seed oil, parsley, sausages) was performed in this study.