We identify the seminal fluid proteins in a monandrous fly, Drosophila subobscura, using a label-free quantitative proteomics approach. The accessory glands and ejaculatory ducts of mated and unmated flies were dissected. Tissues from 10 males were pooled for each sample of mated and unmated males and there were 5 samples per mating treatment. We identified proteins that had significantly higher abundance in unmated than mated males. These proteins were likely to be transferred to females during mating and so represent the candidate seminal fluid proteins.