In vitro fertilization of oceanic squid is a necessary step to develop their larval culture and creates new opportunities to study and understand cephalopod development, taxonomy and ecology. The techniques described here in the form of a laboratory guide represent an attempt to refine and standardize the general methodology by indicating suitable laboratory materials, sources and preservation of gametes, and methods for fertilization and egg incubation. Twelve oceanic squid species have been fertilized in vitro to date; we outline a generalized experimental protocol and suggest that the reader consider particular species-specific modifications. Inadequate egg chorion expansion and premature hatching are identified as major challenges for in vitro fertilization. Recommendations for future research include studies on optimal gamete concentration, gamete preservation and determination of the functions of female oviducal and nidamental glands. The greatest obstacles to improving fertilization success in squids are the lack of standard methodologies and the paucity of information on both endogenous and exogenous factors controlling the fertilization process. This review is a first step toward overcoming these challenges. © 2012 Elsevier B.V.