It was recently suggested that applying high-frequency short biphasic pulses (HF-IRE) reduces pain and muscle contractions in electrochemotherapy and irreversible ablation treatments; however, higher amplitudes with HF-IRE pulses are required to achieve a similar effect as with monophasic pulses. HF-IRE pulses are in the range of a microseconds, thus, the so-called cancellation effect could be responsible for the need to apply pulses of higher amplitudes. In cancellation effect, the effect of first pulse is reduced by the second pulse of opposite polarity. We evaluated cancellation effect with high-frequency biphasic pulses on CHO-K1 in different electroporation buffers. We applied eight bursts of 1 – 10 µs long pulses with inter-phase delays of 0.5 µs – 10 ms and evaluated membrane permeability and cell survival. In permeability experiments, cancellation effect was not observed in low-conductivity buffer. Cancellation effect was, however, observed in treatments with high-frequency biphasic pulses looking at survival in all of the tested electroporation buffers. In general, cancellation effect depended on inter-phase delay as well as on pulse duration, i.e. longer pulses and longer interphase delay cause less pronounced cancellation effect. Cancellation effect could be partially explained by the assisted discharge and not by the hyperpolarization by the chloride channels.