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Abstract BackgroundIncentive salience processes are important for the development and maintenance of addiction. Eye characteristics such as gaze fixation time, pupil diameter, and spontaneous eyeblink rate (EBR) are theorized to reflect incentive salience and may serve as useful biomarkers. However, conventional cue exposure paradigms have limitations that may impede accurate assessment of these markers. ObjectiveThis study sought to evaluate the validity of these eye-tracking metrics as indicators of incentive salience within a virtual reality (VR) environment replicating real-world situations of nicotine and tobacco product (NTP) use. MethodsNTP users from the community were recruited and grouped by NTP use patterns: nondaily (n=33) and daily (n=75) use. Participants underwent the NTP cue VR paradigm and completed measures of nicotine craving, NTP use history, and VR-related assessments. Eye-gaze fixation time (attentional bias) and pupillometry in response to NTP versus control cues and EBR during the active and neutral VR scenes were recorded and analyzed using ANOVA and analysis of covariance models. ResultsGreater subjective craving, as measured by the Tobacco Craving Questionnaire–Short Form, following active versus neutral scenes was observed (F1,106PF1,106PF1,102P ConclusionsThis study provides additional evidence for attentional bias, as measured via eye-gaze fixation time, and pupillometry as useful biomarkers of incentive salience, and partially supports theories suggesting that incentive salience diminishes as nicotine dependence severity increases.