Key enzymes that assemble the bacterial cell wall are also the target of the Β-lactam class of antibiotics. The covalent binding of labeled penicillin to these proteins has been used in numerous studies in drug discovery, antibiotic mechanisms of action and resistance, and cell wall physiology. Methods to label and measure penicillin binding proteins in two prototypical organisms, a Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus), are described. The methods discussed include identifying penicillin-binding proteins in both intact cells (in vivo measurements) and isolated cell membranes.