Infrared ion spectroscopy (IRIS) can be used to identify molecular structures detected in mass spectrometry (MS) experiments and has potential applications in a wide range of analytical fields. However, MS-based approaches are often combined with orthogonal separation techniques, in many cases liquid chromatography (LC). The direct coupling of LC and IRIS is challenging due to the mismatching timescales of the two technologies: an IRIS experiment typically takes several minutes, whereas an LC fraction typically elutes in several seconds. To resolve this discrepancy, we present a heartcutting LC-IRIS approach using a setup consisting of two switching valves and two sample loops as an alternative to direct online LC-IRIS coupling. We show that this automated setup enables us to record multiple IR spectra for two LC-features from a single injection without degrading the LC-separation performance. We demonstrate the setup for application in drug metabolism research by recording six m/z-selective IR spectra for two drug metabolites from a single 2 μL sample of cell incubation extract. Additionally, we measure the IR spectra of two closely eluting diastereomeric biomarkers for the inborn error of metabolism pyridoxine-dependent epilepsy (PDE-ALDH7A1), which shows that the heartcutting LC-IRIS setup has good sensitivity (requiring ∼μL injections of ∼μM samples) and that the separation between closely eluting isomers is maintained. We envision applications in a range of research fields, where the identification of molecular structures detected by LC-MS is required.