The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.