The honeybee has an invaluable economic impact and is a model for studying immunity, development and social behavior. The recent sequencing and annotation of the honeybee genome facilitates the study of its hemolymph, which reflects the physiological condition and mediates immune responses. We aimed at making a proteomic reference map of honeybee hemolymph and compared gel-free and gel-based techniques. One hundred and four 2-DE spots corresponding to 62 different proteins were identified. Eight identical 2-DLC experiments resulted in the identification of 32 unique proteins. One repeat was clearly not representative for the potential of the given 2-DLC setup. Only 27% of the identified hemolymph proteins were found by both techniques. In addition, we found proteins of three different viruses which creates possibilities for biomarker design. Future hemolymph studies will benefit from this work.