Abstract: Microbial inorganic nitrogen (N) immobilization is an important mechanism in the retention of N in soils. Due to the high diversity and complexity of soil microorganisms, how to distinguish and quantify the inorganic N immobilization by fungi and bacteria is a challenging issue. The amino sugar-based stable isotope probing (AS-SIP) approach has been used to indicate the respective inorganic N immobilization rates of fungal and bacterial communities in soils. The effects of agricultural land-use and exogenous organic carbon input on the respective fungal and bacterial nitrate immobilization in soil were unraveled for the first time. Furthermore, a mathematical framework, combining the experimentally measurable gross inorganic N immobilization rate and proxies for fungal and bacterial inorganic N immobilization rates, was proposed to quantify the respective immobilization rates of inorganic N by fungal and bacterial communities in soil. This paper introduces the principle, advantages, case studies, limitations, and coping strategies of this novel AS-SIP approach, aiming to promote its development and application.