Pneumatically assisted electrospray mass spectrometry of polyether ionophores yields several molecular ions. A single metal adduct molecular ion can be obtained by the addition of a neutral salt to the HPLC mobile phase. This approach may be useful in structural studies of unknown ionophores and in the development of specific methods for their analysis in complex matrices. Collision-induced dissociation of the molecular ions provides additional structural information and enhanced specificity for trace analysis. HPLC mobile-phase composition and flow rates have been optimized for on-line analysis. Best response and lowest background noise were obtained at the flow rate of 40 microL/min of a mobile phase containing a 20/80 mixture of water and acetonitrile. The development of a specific confirmatory assay for the new ionophore semduramicin in chicken liver demonstrates the usefulness of on-line HPLC pneumatically assisted electrospray mass spectrometry.