Lateral flow immunoassays (LFA) are one of the most prevalent point-of-care (POC) diagnostics due to their simplicity, low cost, and robust operation. A common criticism of LFA tests is that they have poor detection limits compared to analytical techniques, like ELISA, which confines their application as a diagnostic tool. The low detection limit of LFA and associated long equilibration times is due to kinetically limited surface reactions that result from low target concentration. Here we use isotachophoresis (ITP), a powerful electrokinetic preconcentration and separation technique, to focus target analytes into a thin band and transport them to the LFA capture line resulting is a dramatic increase in the surface reaction rate and equilibrium binding. We show that ITP is able to improve limit of detection (LoD) of LFA by 400-fold for 90 second assay time and by 160-fold for a longer 5 minutes time scale. ITP-enhanced LFA (ITP-LF) also shows up to 30% target extraction from 100 ┬ÁL of the sample, while conventional LFA captures less than 1% of the target. ITP improves LoD of LFA to the level of some lab based immunoassays, such as ELISA, and may provide sufficient analytical sensitivity for application to a broader range of analytes and diseases that require higher sensitivity and lower detection limits.