Microinjection is a method commonly used to deliver various substances into cells. The procedure is performed on a widefield microscope stage using fine glass needle to penetrate the cell membrane. Microinjection can be carried out using a manual or semi-automatic mode. For commercially available equipment currently reported microinjection success rate and cell viability are relatively low (around 50% for both indicators). Here, for the first time, we systematically show how the microinjection effectiveness and cell viability are influenced by needle diameter and chosen microinjection mode. We found that manual mode entailed a higher injection rate, reducing cell viability at the same time. The reduction in needle diameter caused a significant increase in cell survival rate and a slight decrease in success rate for both modes. Our findings will help optimize this method in the context of cell biology research.