We investigate the effect of chip tip-to-counter electrode spacing (gap), channel-size aspect ratio, onset voltage on electrospray ionization (ESI) performance of embossed polymer microfluidic chips. Planar polymer substrates were hot embossed using an electroformed tool and a laser machined tool. The embossed microchips were tested for successful ESI demonstration with minimum sample consumption and high throughput. Factors influencing the stability of electrospray were analyzed by a series of experiments. Theoretically and experimentally, it was observed that the distance of the microchannel tip to the counter electrode directly relates to the onset voltage applied. Furthermore, the overall pattern observed is the decrease in Taylor cone size with a decrease in channel dimensions. The total ion current was analyzed as a function of time and onset voltage at various gaps. Five electrospray modes, namely, drop mode, pulse mode, Taylor-cone jet mode, multijet mode, and oscillating jet mode recorded at the open channel tips were identified and the electrospray cycle investigated. These results are in good accordance with theory, and comparisons are drawn with the findings of other researchers.