The growing emphasis on including invertebrates in global biodiversity conservation efforts has prompted an increase in the study of invertebrate assemblages. Invertebrate sampling designs and the bias of individual methods, nevertheless, remain poorly understood for a variety of habitats. We used a structured inventory approach to sampling ants in five upland ecosystems in Florida. We evaluated the efficiency of quantitative and nonquantitative methods for sampling ants. We also evaluated the performance of four species richness estimators. A total of 3,774 species occurrences were distributed among 1,732 samples that contained 94 species from 31 genera. Twenty unique species and 10 duplicate species were collected. Compared with a comprehensive species list for Florida, sampling captured ≈66% of the regional fauna and ≈70–90% of species within the ecosystems studied. Combinations of sampling methods were much more effective for assessing species richness. Individual methods were complementary and sampled only part of the entire assemblage. Nonparametric estimators (an incidence-based coverage estimator [ICE] and a jackknife estimator [Jack2]) performed better than lognormal fitting, and Michaelis-Menten curve extrapolation. However, none of the estimators was stable, and their estimates should be viewed with skepticism. The results of this study provide support for the use of the Ants of the Leaf Litter (ALL) protocol for thoroughly sampling ant assemblages in temperate and subtropical ecosystems. Furthermore, our results indicate that even in relatively species-poor (compared with the tropics) temperate and subtropical regions, a large sampling effort that includes multiple sampling methods is the most effective manner of thoroughly sampling an ant assemblage. Therefore, we suggest that structured inventory should be adopted for a wider variety of terrestrial invertebrate studies.