Biosolids land application is governed by N content and estimates of potentially available N (PAN). Amino sugar test N (AST-N) has been used with varying success to estimate soil responsiveness to N and optimum N rates. We investigated the utility of an amino sugar test (AST) in estimating PAN and hypothesized that this would depend on biosolids type, rate, and receiving soil. In vitro, we applied three dissimilar biosolids at five rates to four representative southeastern US soils, measured AST-N, and estimated recovery of biosolids AST-N. Target PAN rates were zero to two times a realistic yield expectation rate (127 kg N ha−1) for a common biosolids-receiving grass. Rates were based on biosolids type, total N, and book-value availability coefficients. Biosolids AST-N varied from 263 to 9790 mg kg−1 (3.8–20.1% of total N). Soil AST-N was 66–93 mg kg−1 and differed among soils. Treatment interactions indicated that AST-N of the biosolids–soil mixtures differed from what might be predicted from biosolids and soil AST-N and rate. Rate response was linear; thus, the AST did not saturate at the rates tested. Biosolids AST-N recovery ranged from −303 to 152% depending on biosolids, rate, soil, and their interactions. The AST-N was related linearly to total N from anaerobic incubation (R2 = 0.10–0.67), depending on biosolids. The weakness of these relationships; the biosolids, rate, and soil interactions; and the potential confounding effects of biosolids and soil NH4–N suggest that AST-N would not be a good estimator of PAN.