A reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the content determination of IR780-oleyl (IRO) dye in lipid nanoparticles was developed and validated. Chromatographic separation was performed on a RP C18 column with a gradient program of water and acetonitrile both with 0.1% (v/v) TFA, at a flow rate of 1.0 mL/min and a total run of 21 min. IRO dye detection was made by fluorescence at emission wavelength of 773 nm (excitation wavelength: 744 nm). According to ICH guidelines, the developed method was shown to be specific, linear in the range 3–8 ?g/mL (R2 = 0.9998), precise at the intra-day and inter-day levels as reflected by the coefficient of variation (CV ? 1.98%) at three different concentrations (4, 6 and 8 ?g/mL) and accurate, with recovery rates between 98.2–101.6% and 99.2–100.5%. The detection and quantitation limits were 0.41 and 1.24 ?g/mL, respectively. Stability studies of sample processing showed that IRO dye was stable after 24 h in the autosampler or after three freeze/thaw cycles. Combined with fluorescence measurements, the developed method was successfully applied to optimize the loading capacity of IRO dye in the core of lipid nanoparticles. © 2017 Elsevier B.V.