Using primary neuronal cell culture assays, combined with 2-D gel electrophoresis and capillary LC-MS, we identified differences in proteomes between control and morphine-treated cells. Statistically significant differences were observed among 26 proteins. Nineteen of them were up-regulated, while seven were down-regulated in morphine-treated cell populations. The identified proteins belong to classes involved in energy metabolism, associated with oxidative stress, linked with protein biosynthesis, cytoskeletal ones, and chaperones. The detected proteins demand further detailed studies of their biological roles in morphine addiction. It is crucial to confirm observed processes in vivo in order to reveal the nature and importance of the biological effect of proteome changes after morphine administration. Further investigations may lead to the discovery of new proteome-based effects of morphine on living organisms.