Developing an efficient and effective protocol for capturing biological data held in natural history collections is critically important for many emergent projects in biodiversity, such as the construction of a validated, global DNA barcode reference library. To this end, we developed and streamlined a workflow for ‘museum harvesting’ of authoritatively identified Diptera specimens from the Smithsonian National Museum of Natural History (USNM). Our detailed workflow includes both on-site and off-site processing through specimen selection, labeling, imaging, tissue sampling, databasing and DNA barcoding. This approach was tested by harvesting and DNA barcoding 941 voucher specimens, representing 32 families, 819 genera, and 695 identified species collected from 100 countries. We recovered 867 sequences (> 0 base pairs) with a sequencing success of 88.8% (727 of 819 sequenced genera gained a barcode > 300 base pairs). While Sanger-based methods were more effective for recently-collected specimens, the methods employing next-generation sequencing recovered barcodes for specimens over a century old. The utility of the newly generated reference barcodes is demonstrated by the subsequent taxonomic assignment of nearly 5000 specimen records in the Barcode of Life Data System.