The goal of this work was to present two high-performance liquid chromatography (HPLC) method that could be applied for the determination of the total radioactive purity of 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET). The separation of [18F]fluoride ions, [18F]FET and [18F]FET intermediate was accomplished on LiChrosper RP-18, 250 × 4 mm, 5 µm (Merck) analytical column. For mobile phase 10 mM potassium dihydrogen phosphate buffer at pH7 (A) and acetonitrile (B) was used: 0–2 min: 15% B; 2–12 min: 85% B; 12–15 min: 15% B, respectively. Analysis of [18F]FDG was performed using LiChrosper 100 NH2, 250 × 4.5 mm, 5 µm (Merck) analytical column. The initial mobile phase composition was 10 mM KH2PO4 buffer (pH7) and acetonitrile (15:85, v/v) and the acetonitrile ratio was decreased to 15% at 2 min after the sample injection and held for 5 min. Complete elution of [18F]fluoride ions from stationary phases could be achieved by adding 10 mg/mL K[19F]F to radioactive samples in a ratio 1:1 during the sample preparation. Recovery of [18F]fluoride ions ranged from 99.5 to 100.6%. The validation of the developed methods showed good results for linearity (r2 = 0.9981–0.9996), specificity (RS = 3.7–10.2), repeatability (%Area RSD% = 1.2–4.3%) and limit of quantitation (LOQ = 1.6–4.5 kBq). During the cross-validation similar radiochemical purity values were obtained by the novel HPLC methods and thin layer chromatography performed according to the recommendations of the Ph. Eur. monographs.