We have devised a protocol for highly efficient and specific knockdown of proteins in neuronal cultures. Small hairpin RNAs (shRNAs) are embedded into a microRNA (miRNA) context by oligo annealing to create shRNAmiRs, which are expressed from within the 3'-UTR of a reporter protein. This reporter protein/synthetic miRNA cassette is transferred to a targeting vector and lentivirus is produced in HEK-293-T cells following co-transfection of the targeting vector with three additional vectors encoding essential lentiviral proteins. Mature virus is harvested by collecting culture medium from transfected HEK-293-T cells, the virus is purified by centrifugation, and virus titers are determined prior to addition to neuronal cultures. Near 100% transduction efficiency of cultured hippocampal neurons is routinely observed and allows for the population-wide inhibition of target protein expression and the simultaneous knockdown of multiple proteins with little or no toxicity. The lentivirus generated can be used for protein knockdown in multiple neuronal culture models and at a variety of developmental stages. The steps from shRNAmiR design to ready-to-use virus stocks can be completed in as little as two weeks.