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ABSTRACTBackground Standard methods assessing pain in rodents are often observer dependent, potentially resulting in biased outcomes. Advanced dynamic weight bearing (ADWB) offers an observer-independent approach that can provide objective, reliable data in preclinical pain research.Aims The aim of this study was to characterize the use of ADWB in assessing murine responses to allyl isothiocyanate (AITC)-induced hyperacute hypersensitivity and identify best practices for use of the device.Methods Male C57BL/6J mice received intraplantar injections of saline or 0.1% AITC solution and were assessed using the ADWB system; simultaneous observer-dependent durations of paw licking and biting were measured. ADWB data were analyzed using the proprietary software from Bioseb and correlated to observer-dependent results, with parameters assessed to optimize data collected.Results ADWB detected pain-directed changes in weight and surface area distribution in AITC-treated mice, with paw weight and surface area placement correlating to paw licking and biting. Optimization of adjustable threshold parameters allowed for reduced coefficients of variability and increased duration of validated data.Conclusions The ADWB assay provides an efficient and unbiased measure of chemical-induced hyperacute hypersensitivity in mice. ADWB detection parameters influence amount of validated data and variability, a consideration for data analysis in future studies.