During fear conditioning, pupil size responses dissociate between conditioned stimuli that are contingently paired (CS+) with an aversive unconditioned stimulus, and those that are unpaired (CS-). Current approaches to assess fear learning from pupil responses rely on ad hoc specifications. Here, we sought to develop a psychophysiological model (PsPM) in which pupil responses are characterized by response functions within the framework of a linear time-invariant system. This PsPM can be written as a general linear model, which is inverted to yield amplitude estimates of the eliciting process in the central nervous system. We first characterized fear-conditioned pupil size responses based on an experiment with auditory CS. PsPM-based parameter estimates distinguished CS+/CS- better than, or on par with, two commonly used methods (peak scoring, area under the curve). We validated this PsPM in four independent experiments with auditory, visual, and somatosensory CS, as well as short (3.5 s) and medium (6 s) CS/US intervals. Overall, the new PsPM provided equal or decisively better differentiation of CS+/CS- than the two alternative methods and was never decisively worse. We further compared pupil responses with concurrently measured skin conductance and heart period responses. Finally, we used our previously developed luminance-related pupil responses to infer the timing of the likely neural input into the pupillary system. Overall, we establish a new PsPM to assess fear conditioning based on pupil responses. The model has a potential to provide higher statistical sensitivity, can be applied to other conditioning paradigms in humans, and may be easily extended to nonhuman mammals.