The appropriate concentration of antibiotic for selecting stable cell lines is different for each cell type. If the concentration for the desired cell type is unknown, a titration experiment must be performed to determine the lowest concentration of antibiotic needed to efficiently select transduced cells. In this protocol, we highlight the use of puromycin, the antibiotic used with the standard pLKO.1 vector. Typically, 1-10 µg/mL are sufficient to kill most untransduced mammalian cell types. This general protocol can also be used for determining the optimal concentration of G418 required for the custom shRNA vectors. Higher antibiotic concentrations than required could result in off-target effects and fewer cells for downstream analysis.