Our overall goal is to apply acoustic separation technology for the recovery of valuable particulate matter from wastewater in industry. Such large-scale separator systems require detailed design and evaluation to optimize the system performance at the earliest stage possible. Numerical models can facilitate and accelerate the design of this application; therefore, a finite element (FE) model of an ultrasonic particle separator is a prerequisite. In our application, the particle separator consists of a glass resonator chamber with a piezoelectric transducer attached to the glass by means of epoxy adhesive. Separation occurs most efficiently when the system is operated at its main eigenfrequency. The goal of the paper is to calibrate and validate a model of a demonstrator ultrasonic separator, preserving known physical parameters and estimating the remaining unknown or less-certain parameters to allow extrapolation of the model beyond the measured system. A two-step approach was applied to obtain a validated model of the separator. The first step involved the calibration of the piezoelectric transducer. The second step, the subject of this paper, involves the calibration and validation of the entire separator using nonlinear optimization techniques. The results show that the approach lead to a fully calibrated 2-D model of the empty separator, which was validated with experiments on a filled separator chamber. The large sensitivity of the separator to small variations indicated that such a system should either be made and operated within tight specifications to obtain the required performance or the operation of the system should be adaptable to cope with a slightly off-spec system, requiring a feedback controller.