Background <p>This study investigated the effects of temperature and concentration on the short-term stability of colistin and colistin methanesulphonate in three biological matrices; whole blood, plasma and serum. </p> Methods <p>Human whole blood, plasma or serum containing colistin (2; 10; 20 mg/L) or colistin methanesulphonate (2; 10; 20 mg/L) was stored at room temperatrure or at 37 °C for a predetermined time interval. The concentrations of colistin in colistin spiked samples, and formed colistin in colistin methanesulphonate spiked samples, were analysed by LC-MS method.</p> Results <p>Findings indicate that colistin A and B were stable at the room temperature, but degradation accelerated at 37 °C, particularly at lower concentrations. The conversion of CMS to colistin was also temperature-dependent, where the concersion was significantly accelerated at elevated temperatures. Freeze-thaw stability were tested, COL A as well as COL B were stable for at least three freeze-thaw cycles.</p> Conclusion <p>These results underscore the importance of rapid sample processing to ensure reliable therapeutic drug monitoring and emphasize the importance of considering temperature and concentration factors in clinical practice to optimise dosing and minimise toxicity. Further research is required to investigate the full range of factors affecting the stability of colistin and colistin methanesulphonate in biological matrices.</p>