Monochromatic excitation X-ray uorescence (ME-XRF) spectrometry is a novel technique for trace element analysis, characterized by its simplicity, rapidity, and low cost. The objective of this study was to evaluate the applicability of ME-XRF technique for the measurement of thallium in biological samples.Acute and subacute thallium poisoning experiments were conducted to simulate various scenarios, with blood, urine, and 10 distinct organs collected. Detection was initially performed using ME-XRF technique, followed by validation with inductively coupled plasma mass spectrometry (ICP-MS). Excellent agreement between ME-XRF and ICP-MS values was demonstrated by means of paired sample t-tests and intraclass correlation coe cients. Subsequently, the practical implementation of the proposed technique was demonstrated through an actual case study. In conclusion, this study validates ME-XRF as a suitable alternative to ICP-MS for the measurement of trace heavy metals in biological samples. These efforts promote the development of simpler and faster techniques for heavy metal detection, thereby presenting novel avenues for the prevention and diagnosis of heavy metal poisoning.