Technical Abstract: The sedentary semi-endoparasitic nematode Rotylenchulus reniformis, the reniform nematode, is a serious pest of cotton and soybean in the United States. In recent years, interest in the molecular biology of the interaction between R. reniformis and its plant hosts has increased; however, the unusual lifecycle of R. reniformis presents a unique set of challenges to researchers who wish to study the developmental expression of a particular nematode gene or evaluate life stage-specific effects of a specific treatment such as RNA-interference or a potential nematicide. Unlike other sedentary plant-parasitic nematodes, the R. reniformis juvenile stages do not feed; only the vermiform adult female infects the host root. In this report, we describe a simple method that allows the collection of R. reniformis juvenile and vermiform adult life stages under in vitro conditions. R. reniformis eggs were hatched over a Baermann funnel and the resultant second-stage juveniles incubated in petri-plates at 30°C. R. reniformis juvenile development was monitored over time and the presence of residual intact juvenile cuticles was used to identify the third-stage and fourth-stage juveniles at specific time-points. Parasitic sedentary females were collected from roots of infected host plants by a combination of blending, sieving, and sucrose floatation. The methods presented here provide a way to collect large quantities of R.reniformis life stages that can be used for nucleic acid or protein extraction or for other experimental purposes.