Purpose: IR780 iodide, a promising near-infrared dye, is widely used to prepare nanoparticles as a theranostic agent for tumor imaging and therapy. However, there are no validated (bio)analytical methods to measure IR780 in nanoparticles and tissues in literature. The aim of this study is to develop and validate a new HPLC method to measure IR780 concentration in IR780 formulations as well as a new LC–MS/MS method to measure IR780 concentration in tissue samples, particularly in liver and lung. Materials and methods: IR780 granules that produced IR780 in situ self-assembled nanoparticles upon contact with water were prepared at two drug loadings (0.2 % and 0.37 %). An HPLC method was developed and validated to measure IR780 concentrations in IR780 granules and nanoparticles. Furthermore, a validated LC–MS/MS method was developed to measure IR780 in mouse liver and lung. Both HPLC method and LC–MS/MS method were validated in terms of specificity, stability, linearity, limit of detection, limit of quantification, accuracy and precision. Results: Both HPLC method and LC–MS/MS method achieved the criteria for method validation. The HPLC method was accurate in the concentration range of 0.5−25 μg/mL. The measured drug loadings were 95 % of the theoretical drug loadings. The validated LC–MS/MS method can quantitatively measure the concentrations of IR780 in liver and lung. The linear range of the LC–MS/MS method was 1−1000 ng/mL for both liver and lung samples. IR780 granules showed the lung selectivity compared to IR780 solution at 2 h after oral administration. Conclusion: A validated HPLC method was developed to measure IR780 concentration in pharmaceutical formulations and a validated LC–MS/MS method was developed to measure IR780 concentration in tissues. These quantitative methods provide reliable measurements of IR780 in pharmaceutic formulations and biological samples, which will significantly facilitate the research of IR780 as a theranostic agent for cancer therapy and imaging.