Information on chemical speciation is much needed in mechanistic and kinetic studies on catalyst formation processes in pharmaceutical research. Speciation analysis was applied to the identification and quantification of various rhodium species involved in a ligand exchange process leading to formation of catalyst dirhodium(II) tetrakis[methyl 2-oxopyrrolidin-5(S)-carboxylate]. Inductively coupled plasma mass spectrometry (ICPMS) was used as an element-specific detector following species separation by reversed-phase high-performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI-MS) was used for species identification and confirmation. A novel interface between the HPLC and ICPMS, which consisted of an eluent splitter, a desolvation unit, and the ICPMS built-in peristaltic pump, enabled the use of RP-HPLC with gradient elution and up to 100% organic components in the LC eluent without organic loading in the plasma. A variety of reaction intermediates were identified and quantified along the pathway to formation of the desired product, including isomeric di-, tri-, and tetrasubstituted species previously believed to be absent. This has provided new insights into the mechanism and kinetics of the reaction. The combination of HPLC-ICPMS and HPLC-ESI-MS has proven to be a valuable tool for the investigation of species evolution in catalyst formation process.