High-frequency bipolar electric pulses have been shown to mitigate undesirable muscle contraction during irreversible electroporation (IRE) therapy. Here, we evaluate the potential applicability of such pulses for introducing exogenous molecules into cells, such as in electrochemotherapy (ECT). For this purpose we develop a method for calculating the time course of the effective permeability of an electroporated cell membrane based on real-time imaging of propidium transport into single cells that allows a quantitative comparison between different pulsing schemes. We calculate the effective permeability for several pulsed electric field treatments including trains of 100μs monopolar pulses, conventionally used in IRE and ECT, and pulse trains containing bursts or evenly-spaced 1μs bipolar pulses. We show that shorter bipolar pulses induce lower effective membrane permeability than longer monopolar pulses with equivalent treatment times. This lower efficiency can be attributed to incomplete membrane charging. Nevertheless, bipolar pulses could be used for increasing the uptake of small molecules into cells more symmetrically, but at the expense of higher applied voltages. These data indicate that high-frequency bipolar bursts of electrical pulses may be designed to electroporate cells as effectively as and more homogeneously than conventional monopolar pulses. Display omitted •Real-time imaging and permeability analysis were performed post-electroporation treatment.•Rapid bipolar electrical pulses induce lower membrane permeability than longer monopolar pulses.•Transmembrane transfer of molecules may be achieved using trains of short pulse widths.•Rapid bipolar pulses need larger amplitudes to disrupt cells similar to longer monopolar pulses.