An improved in vitro rearing system, based on a silicone-reinforced Parafilm® M membrane, human hair tufts and reconstituted human blood, enabled the large-scale rearing of pediculicide-susceptible (EC-HL) and resistant (SF-HL and BR-HL) strains of human head lice. Developmental time differed for early instars but differences became synchronized as lice matured. Mean survivorship amongst the three strains reared in vitro were not significantly different when compared to EC-HL and SF-HL reared in vivo. The efficacies of three pediculicidal products were assessed under semi-clinical conditions using a modified version of the in vitro rearing system. Treatments of 1% permethrin in acetone, Nix®, Rid® or Pronto® Plus to hair tufts following manufacturer’s instructions were highly efficacious (100% mortality) on EC-HL but differentially efficacious (62.2–84.4% mortality) on SF-HL examined eight days post-treatment. SF-HL that survived the first treatment received an identical second treatment eight days following the first treatment. Survivors (13.3–30.0%) developed to adults (10th–11th day following first treatment) and females successfully laid fertile eggs that developed to first instars. These results confirm resistance to permethrin- and pyrethrin-based pediculicide formulations in SF-HL when assessed under semi-clinical conditions and validates resistance previously determined using filter-paper contact bioassays and unformulated insecticides.