Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae . After the successful in vitro production of S. jeffreyense -inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml −1 after 14 days, in comparison to 75% and 99,875 IJs ml −1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml −1 ), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.