In order to achieve a more efficient means of promoting ovarian maturation and spawning of the Pacific white shrimp Litopenaeus vannamei, we evaluated the reproductive performance of female broodstock subjected to unilateral eyestalk ablation compared to that of non-ablated individuals under laboratory conditions. Two consecutive experiments were conducted for a period of eight weeks each, and in both experiments, animals were fed at a rate of 10% of total biomass in each tank. In Experiment I, with the aim of determining an appropriate feeding regimen suitable for re-circulating systems, animals were fed with two dietary regimens: commercial formulated pellets only, and a combination diet of formulated pellets and fresh polychaetes (Nereis sp.). In terms of the incidence of gonadal maturation and number of spawned eggs, unilaterally-ablated shrimp fed the combination diet showed the most satisfactory results. Therefore, in Experiment II, we utilized the same treatments in order to demonstrate the validity of this methodology. The results demonstrated that L. vannamei broodstock can be readily matured and spawned under laboratory conditions, but that it is necessary to include live feeds in the diet in order to achieve a higher rate of maturation and spawning.