A sandwich-type enzyme-linked immunosorbent assay (ELISA) was established for monitoring the secretion of ZZ-fusion proteins. Two antibodies, a monoclonal mouse anti-human proinsulin and a rabbit anti-bovine IgG (strongly binding to the ZZ-domain), were used to quantify the secretion of recombinant human ZZ-proinsulin to the growth medium of Escherichia coli cultures. The method here reported conjugates the advantages of sandwich-type ELISA assays, namely, high sensitivity, specificity, and throughput, with the possibility of quantifying small protein molecules (e.g., peptides). A further advantage of gene fusion techniques integrating both downstream processing and product detection and quantitation is highlighted. The method is capable of detecting levels of 0.05 ng of ZZ-proinsulin.