Concentration polarization (CP)-based focusing electrokinetics nanofluidic devices have been developed in order to simultaneously detect and enrich highly diluted analytes on-a-chip. However, stabilization of focal points over long time under the application of the electric field remains as a technical bottleneck. If pressure-assisted preconcentration methods have been proposed to stabilize propagating modes at low inverse Dukhin number(1/DuMUCH LESS-THAN1), these recent protocols remain laborious for optimizing experimental parameters. In this paper, "electric field E/counter-pressure P" diagrams have been established during pressure-assisted electro-preconcentration of fluorescein as a model molecule. Such E/P diagram allows direct observation of the region for which the optimal counter-pressure P leads to a stable focusing regime. This region of stable focusing is shown to vary depending of the nanoslit length (100 mu m < L-nanoslit< 500 mu m) and the nature of the background electrolyte (KCl and NaCl). Longer nanoslits (500 mu m) produce stabilization at low counter-pressure P, whereas NaCl offers a narrower region of stable focusing in the E/P diagram compared to KCl. Finally, the ability of such pressure-assisted protocol to concentrate negatively charged proteins has been tested with a more applicative protein, i.e., ovalbumin. The corresponding E/P diagram confirms the existence of the stable focusing regime at both low electric field E (<= 20 V) and counter-pressure P (<= 0.4 bar). With an enrichment factor as high as 70 after 2 min for ovalbumin at a concentration of 10 mu M, such pressure-assisted nanofluidic electro-preconcentration protocol appears very promising to concentrate and detect biomolecules.