Fungi are critical components of the soil food web. Fungi are important as organic matter decomposers, nutrient recyclers, and fungal hyphae play a role on the formation of soil aggregates that can increase water infiltration, improve water holding capacity, and sequester soil carbon (C). Ergosterol is a sterol found ubiquitously in cell membranes of filamentous fungi and is commonly used as a marker compound for fungal biomass. In contrast, the analysis of chitin is potentially effective way to monitor changes in total fungal mass (biomass and necromass) under different environmental conditions, because chitinis a more stable compound than ergosterol. By combining this analysis with our previously developed ergosterol method, we can determine both live fungal biomass and by subtraction, fungal necromass, thus providing useful information on the turnover dynamics of total fungal mass. We developed a sample preparation method based on extraction and conversion of chitin to chitosan, after which chitosan is measured by Asymmetrical Flow Field-Flow Fractionation (AF4). Our results show that this analytical method is a simple, fast, and cost-effective technique for the quantitative analysis of chitin from field-collected soils. The detection limit of this method is 6 μg g^–1 chitin (chitosan) in dry soil. The final method linear range is 20 – 500 μg g^–1 for chitin and 0.4 - 10 mg g^–1 for fungal biomass. The developed chitin based and existing ergosterol based fungal estimation methods were used for the soil analysis. These methods can combine to determine the living and total fungal biomass.