Gradient high-performance liquid chromatography (HPLC) preceded by a solid-phase extraction (SPE) step is described for determining arginine vasotocin and isotocin, the neurohypophysial nonapeptides, in fish plasma samples. The combination of these two methods significantly improves the separation and increases the sensitivity of the assay. The proposed assay may be a useful alternative for analysis of similar nonapeptides in plasma without the use of radioisotopes, while taking into consideration a difference in detection sensitivity.