Despite the widely acknowledged importance of arbuscular mycorrhizal fungi (AMF) in soil ecology, quantifying their biomass and presence in field soils is hindered by tedious techniques. Biochemical markers such as ergosterol, and chitin were considered quick reliable tools in AMF detection however; due to the ubiquitous nature of these compounds their usage is limited. Glomalin related soil protein (GRSP) has proven useful in AMF quantification. GRSP is operationally defined, its identification rests solely on the methods used to extract it from soil (citric acid buffer and autoclaving) and the assays (Bradford/ELISA) utilized to detect it. The current assumption is that all non-heat stable proteins except glomalin are destroyed during the harsh extraction procedure. However, this critical assumption has yet to be thoroughly tested. The purpose of this research was to challenge the GRSP extraction process; determine the accuracy of the Bradford method as an indicator of total GRSP; and provide some assessment of the specificity of the ELISA monoclonal antibody. Two studies were developed: 1) soil samples were spiked with a known quantities of a heat-stable glycoprotein (BSA: bovine serum albumin) and 2) soil samples were supplemented with leaf litter from specific sources. Our results indicated 41-84% of the added BSA still remained in our soils. This suggests that the currently used extraction procedure does not eliminate all non-glomalin heat stable proteins. ELISA cross reactivity against BSA was limited ranging from 3-17%. Our second study showed that additions of leaf litter significantly influenced GRSP extraction and quantification suggesting that plant derived proteins had a similar effect as experiment 1. Litter additions decreased the immunoreactive protein values, suggesting interference with antibody recognition. We conclude that the use of GRSP in the assessment of AMF biomass within field soils is problematic, it may be inappropriate in situations of significant organic matter additions.