Reversed-phase ultra-high-performance liquid chromatography (RP-UHPLC) was used for the separation of recombinant human growth hormone (rhGH) variants. A bridged ethylene hybrid silica C18 column was used at 37°C. The composition and temperature of the mobile phase were optimized for the separation. An isocratic elution, with approximately 46% acetonitrile in 25 mM potassium borate buffer (pH 8.5), was found to give superior selectivity in comparison with commonly used mobile phases. The method separated eight rhGH variants: (i) di-oxy Met14/Met125 sulfoxide, (ii) Met125 sulfoxide, (iii) Met14 sulfoxide, (iv) mono-deamidated (Asn149 → Asp149 or Asn152 → Asp152), (v) di-deamidated (Asn149 → Asp149 and Asn152 → Asp152), (vi) clip (Thr142-Tyr143), (vii) desPhe1 and (viii) trisulfide (Cys182-SSS-Cys189) from each other and from the native rhGH. Characterization of the purified variants was conducted by liquid chromatography-mass spectrometry tryptic mapping. The novel mobile phase, in combination with the UHPLC system, generated a significantly higher resolution than previously reported reversed-phase LC methods, including pharmacopoeal methods, for analyzing rhGH.