Traditionally, electroporation-based therapies such as electrochemotherapy (ECT), gene electrotransfer (GET) and irreversible electroporation (IRE) are performed with different but typical pulse durations—100 microseconds and 1–50 milliseconds. However, recent in vitro studies have shown that ECT, GET and IRE can be achieved with virtually any pulse duration (millisecond, microsecond, nanosecond) and pulse type (monopolar, bipolar-HFIRE), although with different efficiency. In electroporation-based therapies, immune response activation can affect treatment outcome, and the possibility of controlling and predicting immune response could improve the treatment. In this study, we investigated if different pulse durations and pulse types cause different or similar activations of the immune system by assessing DAMP release (ATP, HMGB1, calreticulin). Results show that DAMP release can be different when different pulse durations and pulse types are used. Nanosecond pulses seems to be the most immunogenic, as they can induce the release of all three main DAMP molecules—ATP, HMGB1 and calreticulin. The least immunogenic seem to be millisecond pulses, as only ATP release was detected and even that assumingly occurs due to increased permeability of the cell membrane. Overall, it seems that DAMP release and immune response in electroporation-based therapies can be controlled though pulse duration.