Protein targeting in both eukaryotic and prokaryotic cells is often directed by a signal sequence located at the amino-terminus of the protein. In eukaryotes, proteins that are sorted into different compartments of the cell, such as endoplasmic reticulum, mitochondria, and chloroplast, require different signal sequences. In bacteria, proteins which are exported to the outer membrane or the periplasmic space are also guided by signal peptides. After the protein is translocated across the cytoplasmic membrane, the signal peptide is proteolytically removed by signal peptide cleavage. Here, in this chapter, we describe methods to study signal peptide processing in bacteria, including purification of signal peptidase and its substrates. We also describe the measurement of the catalytic constants of signal peptidases using an in vitro assay. In addition, we will present an in vivo assay using a temperature sensitive signal peptidase strain to determine which preproteins are processed by Signal peptidase 1.