Background/Question/Methods Feeding patterns of generalist predators present intriguing challenges for ecologist due to the complexity of these food webs. Spiders (Araneae) comprise one of the most common predatory groups in terrestrial systems. It is difficult to quantify their feeding frequency, because they use extraoral digestion, and are documented to consume a range of prey types. To explore the influence of seasonality, habitat type and prey availability on consumption patterns, we used a recently developed Diptera specific monoclonal antibody. We selected an epigeal wolf spider, Pardosa milvina (Lycosidae), which is common in agricultural systems to quantify its consumption of a single prey group, Diptera. We collected spiders weekly across the season in corn and soybean fields to compare between these two agricultural field types. Because it is often assumed that generalist predators consume suitable prey in proportion to abundance, in conjunction with screening Pardosa for Diptera, we estimated prey availability in these fields using D-Vac suction sampling. Results/Conclusions Of the more than 1700 spiders analyzed using enzyme-linked immunosorbent assay to detect Diptera proteins, 10% of Pardosa tested positive. Frequency of predation on Diptera varied over the season ranging from less than 10% to more than 30% in a weekly sample. Although there were no distinguishable differences in consumption of Diptera by females as compared to males, juvenile spiders consumed Diptera less frequently than either males or females. Habitat type, either soybean or corn crops, had no observable impact on Diptera consumption. Prey availability varied across the season, but there were no strong correlations with predation frequency. Notably, a shift in the abundance of Collembola, a recognized prey group for this species, did not correspond with a significant shift in predation on Diptera. Consumption frequency of Diptera, in our study, was much less than reported in previous studies on similar species in the genus Pardosa and may indicate higher dietary diversity than previously acknowledged. That Pardosa milvina selected Diptera independent of variation of other prey groups indicates consistent reliance on Diptera that may be related to essential nutrients needed during reproductive activities or potential age specific dietary requirements.