A novel HPLC method was developed and validated to simultaneously determine curcuminoids and dasatinib in a new dosage form. Excellent separation was achieved for a curcuminoids mixture and dasatinib with a ZORBAX Eclipse Plus Phenyl–Hexyl column (4.6 × 250 mm, 5 µm) and a mobile phase consisting of aqueous solution (0.2% acetic acid and 0.1% trifluoroacetic acid with the pH adjusted to 3.5) and acetonitrile with a gradient elution at a flow rate of 1 mL/min. The separated peaks were detected at 325 nm for dasatinib and 420 nm for the curcuminoids mixture. The mixture was separated in less than 20 min. The retention times were 6.5 min, 16.4 min, 17.5 min, and 18.4 min for dasatinib, bisdesmethoxycurcumin, desmethoxycurcumin, and curcumin, respectively. The linear range for all compounds was 0.5–140 µg/mL with a limit of detection of 0.10–0.50 µg/mL. The method was validated according to the International Council for Harmonization guidelines and was shown to be sensitive, fast, robust, specific, and accurate. The developed method was successfully applied to determine dasatinib and curcuminoids in a nanoparticle preparation with good accuracy and precision. The method may be utilized to conduct in vitro studies for new pharmaceutical formulations containing a similar combination of compounds.