An HPLC method is presented for the determination of lanthanides viz. lutetium (Lu), ytterbium (Yb), thulium (Tm), erbium (Er), holmium (Ho), terbium (Tb), and yttrium (Y) in high-purity dysprosium oxide (Dy2O3) matrix. In view of their similar chemical and physical properties, determination of rare-earth elements in bulk of a rare-earth element is a difficult task. Studies were carried out on C18 and C8 reversed phase columns employing α-hydroxyisobutyric acid (α-HIBA) as an eluent. n-Octane was used as an ion interaction reagent (IIR). Different chromatographic parameters e.g., ion interaction reagent, pH and concentration of α-HIBA were studied simultaneously to arrive at the optimum condition for the individual separation of lanthanides. When n-Octane sulphonate was used as the IIR, C8-based stationary phase was found to give significantly better resolution as compared to C18-based stationary phase. Under the optimized conditions, it was possible to resolve Y from Dy using α-HIBA as the single eluent. Separation and quantification of rare-earth elements were possible in samples with Dy/Tb and Dy/Y amount ratios up to 200. The method was validated using the simulated sample containing above lanthanides, Y and Dy matrix. Quantification was done using standard addition method. The method was successfully applied for the quantification of lanthanides and Y in high purity Dy2O3 sample. Concentrations of Lu, Yb, Tm, Er, Ho, Tb, and Y were found to be 74 ppmw, 432 ppmw, 22 ppmw, 57 ppmw, 3325 ppmw, 1207 ppmw, and 4463 ppmw, respectively.