Vitellogenesis in crustaceans is under the inhibitory regulation of the endocrine system in the X-organ/sinus gland complex in the eyestalks. In this study, two monoclonal antibodies specific to Macrobrachium rosenbergii vitellin and vitellogenin were generated to assess vitellogenesis-inhibiting hormone (VIH) activity. A combination of two monoclonal antibodies specific to the 89 and 100 kDa subunits of vitellin were used to develop a competitive enzyme-linked immunosorbent assay protocol for the quantitative determination of vitellogenin levels in haemolymph. Injection of eyestalk extracts from M. rosenbergii dissolved in either saline or in methanol-acetic acid caused a reduction in vitellogenin levels in the haemolymph at 12 and 24 h post-injection in bilateral eye-ablated prawns with developing ovaries. In contrast, the application of the heterologous eyestalk extract from Penaeus vannamei did not reduce vitellogenin levels in haemolymph. This assay is therefore specific and could be used to assess VIH activity during hormone purification.