Pulsed nanoelectrospray ionisation (pulsed nESI) is an emerging technique for ionising volatile biomolecules for sensitive detection by mass spectrometry (MS). In conventional nESI, direct current high voltage (DC HV) is applied to the analyte solution resulting in the formation of ions with high sensitivity, low sample consumption and low limits of detection. Alternatively, in pulsed nESI, molecules are ionized by using a pulsed high voltage square waveform. During the course of this research project, another group reported that pulsed nESI can be used to measure peptide and protein ions by applying short, repetitive pulses of high voltage to the nESI emitter. However, the performance of such an ion source was not compared to conventional nESI. Thus, the extent that pulsed nESI can be used to improve performance of MS was unclear. Here, pulsed nESI is reported in which the voltage is rapidly pulsed from 0 to up to ~3 kV with a rise time of low to sub-nanoseconds and with duty cycles ranging from 1-50% corresponding to pulse durations of 9 to 450 μs. By use of pulsed nESI, the performance of MS for the detection of many different classes of molecules can be improved in terms of decreasing background chemical noise and enhancing the sensitivity for analytes ranging from small molecules to whole proteins. Specifically, pulses that are less than ~450 μs can be used to decrease the background chemical noise and increase signal-to-background chemical noise ratio by up to 93% and 691% in pulsed nESI MS for six test analytes compared to conventional nESI. Pulsed nESI can also be used for native mass spectrometry to improve signal-to- background chemical noise ratios. Overall, pulsed nESI can be used to significantly improve the performance of MS.