Reversed-phase high-performance liquid chromatography (RP-HPLC) and ion-exchange chromatography on poly(2-sulphoethylaspartamide)-silica (SCX) were compared as alternative approaches in characterizing charged genetic variants of human serum albumin. The chromatographic behaviour of cyanogen bromide (CNBr), tryptic and V8 protease digests from normal and mutant albumins were examined. The results showed that substituted site-containing CNBr fragments are successfully resolved by RP-HPLC; in most instances SCX and RP-HPLC are equally adequate in identifying the modified tryptic peptides from CNBr fragments; although generally useful, SCX chromatography is specifically needed in all instances where amino acid replacement is occurring in a small hydrophilic tryptic fragment and choosing Staphylococcus aureus V8 protease instead of tryptic digestion is advantageous.