A semipreparative HPLC setup was evaluated for automated fractionation with both photometric- and mass spectrometric trigger. The goals of the work were to systematically study and optimize the flow-splitting setup for mass-directed purifications by mathematical modeling and experimental verification. The system comprised a passive splitting device with make-up flow capability, which directed a small fraction of the column effluent to the mass spectrometer and the remainder to the fraction collector. Tubing lengths and diameters of the splitter as well as make-up flow rates were varied in order to address and optimize peak dispersion, delay times between mass detector and fraction collector, and mass spectrometric signal quality. A paraben standard mixture was analyzed and purified on both microparticulate and monolithic columns with 10 mm inner diameter and at typical flow rates of 5-10 mL/min. Fraction purities and recoveries close to 100% were achieved. The system allowed mass-triggered fractionations on a 1 mg scale at flow rates of 10 mL/min in combination with monolithic columns in less than 2 min. Finally, the system was successfully applied to the fully automated isolation of milligram quantities of degradation products in a pharmaceutical preparation to successfully allow for structure elucidation with NMR spectroscopy.