In most instances, baseline data for biotic conditions within a pre-determined reference site are collected. Reference sites must be characterized by little to no outside disturbance (e.g. anthropogenic disturbances, land use change, invasive species). The biotic conditions of a specific indicator species are measured within both the reference site and the study region over time. Data collected from the study region are compared against similar data collected from the reference site in order to infer the relative environmental health or integrity of the study region.
An important limitation of bioindicators in general is that they have been reported as inaccurate when applied to geographically and environmentally diverse regions. As a result, researchers who use bioindicators need to consistently ensure that each set of indices is relevant within the environmental conditions they plan to monitor.