The F₂ screen is a flexible methodology used to estimate the frequency of resistance alleles (R) in an insect population. We have developed several alternative protocols, along with the associated statistics, to conduct an F₂ screen with mated or unmated individuals, random and nonrandom mating of F₁ adults, and the screening of multiple lines together in the F₂ screen. Our protocols describe how to perform and analyze an F₂ screen starting with unmated P₁ as an alternative to mated females. A randomly mated population of ≥50 F₁ adults should be sufficient to detect R alleles >99% of the time. If nonrandom mating occurs in the F₂ screen, it is most likely to be positive assortative mating, and this would improve the probability of detecting an R allele. Pair mating the F₁ adults greatly increases costs of the screen while providing a small, but negligible improvement in detecting R alleles. The number of screens may be reduced by more than two-thirds by screening multiple lines together. These methodological variants show the F₂ screen to be much more robust than originally described.