A microelectroporation system was built to provide a well-controlled and defined environment for quantitative studies of electroporation, a method to permeabilize cell membranes for delivery of normally impermeable biomolecules, of an immortalized line of T cells. Studying electroporation in a low ionic-strength solution, onset of poration was found to occur at an externally applied field of 700 V/cm, corresponding to a threshold transmembrane potential of 0.83±0.34 V. A finite element model was used to estimate the effect of solution conductivity on the applied electric field, which dominates the uncertainty in this measurement. Furthermore, increased forward light-scattering signal, as measured using flow cytometry, correlated strongly to successful poration. Using forward light-scattering as an indicator identifies a population of cells that is over 90% porated and viable. Quantitative characterization with flow cytometry could serve as a non-invasive assay of microelectroporation in T-cells that can be applied without fluorescent markers.