Ampicillin is widely used in the treatment of bacterial infections caused by susceptible microorganisms. It is effective against a range of Gram-positive and some Gram-negative bacteria. Common applications include treating respiratory tract infections, urinary tract infections, meningitis, and gastrointestinal infections caused by bacteria such as Escherichia coli, Haemophilus influenzae, and certain strains of Salmonella and Shigella.

In clinical settings, ampicillin is administered orally, intravenously, or intramuscularly depending on the severity of infection. It is often used in combination with other antibiotics, such as sulbactam, to enhance its effectiveness against beta-lactamase-producing bacteria.

In laboratory research, ampicillin is frequently used as a selective agent in molecular biology. It helps maintain plasmid vectors in bacterial cultures, especially in E. coli, by selecting for cells that have taken up antibiotic resistance genes during transformation experiments. This makes it a critical tool in genetic engineering and recombinant DNA technology.