Paper substrates have been widely used to construct point-of-care lateral flow immunoassays (LFIA) diagnostic devices. Paper based microfluidic devices are robust and relatively simple to operate, compared to channel microfluidic devices, which is perhaps their greatest advantage and the reason they have reached a high level of commercial success. However, paper devices may not be well suited for integrated sample preparation, such as sample extraction and preconcentration, that is required in complex samples with low analyte concentrations. In this study, we investigate integration of isotachophoresis (ITP), an electrokinetic preconcentration and extraction technique, onto nitrocellulose-based paper microfluidic devices in the goal to improve the limit of detection of LFIA. ITP has been largely used in traditional capillary based microfluidic devices as a pre-treatment method to preconcentrate and separate variety of ionic compounds. Our findings show that ITP on nitrocellulose is capable of up to 900 fold increase in initial sample concentration and up to 60% extraction from 100 ┬ÁL samples and more than 80% extraction from smaller sample volumes. Paper based ITP is challenged by Joule heating and evaporation because it is open to the environment. We achieved high preconcentration by mitigating evaporation induced dispersion using novel cross-shaped device structures that keep the structures hydrated. We show that ITP on nitrocellulose membrane can be powered and run several times by a small button battery suggesting that it could be integrated to a portable point-of-care diagnostic device. These results highlight the potential of ITP to increase sensitivity of paper based LFIA in conditions where small analyte concentrations are present in complex biological samples.