Entomopathogenic baculoviruses have long been recognized as vital environmentally safe alternatives to chemical pesticides. A study for investigating the virulence and susceptibility of Spodoptera littoralis larvae to S. littoralis nucleopolyhedrovirus (SpliNPV) isolate was carried out. Bioassay experiments on the 1^st and 2^nd larval instars using droplet feeding method revealed that polyhedral for 50% or 90% mortality of the 2^nd larval instar of S. littoralis was significantly lower compared with the 1^st instar. While, the 1^st instar was killed faster than the 2^nd one especially with the higher concentration. The susceptibility of the infected 2^nd instar when feeding on different hosts showed that larvae were more susceptible when feeding on eggplant leaves followed by lettuce leaves, artificial diet and tomato leaves as shown by larval weight and reduction in feeding. The reduction in both larval weight and feeding were significantly higher with 1x10⁷ PIB/ml than 1x10⁴ PIB/ml in the majority of cases. S. littoralis larvae showed more than 2 times of tolerant to the 3^rd passage of SpliNPV on the level of LC₅₀ and more than 18 times tolerant on the level of LC₉₀. The tolerant levels of S. littoralis larvae for the 3^rd passage of virus was associated with more than 20% increase in time taken for kill with no changes in DNA patterns regarding subsequent passages in S. littoralis larvae using a restriction enzyme EcoRI.