Abstract Object localization is an essential step in image-based hardware assurance applications to navigate the view to the target location. Existing localization methods are well-developed for applications in many other research fields; however, limited study has been conducted to explore an accurate yet efficient solution in hardware assurance domain. To this end, this paper discusses the challenges of leveraging existing object localization methods from three aspects using the example scenario of IC Trojan detection and proposes a novel knowledge-based object localization method. The proposed method is inspired by the 2D string search algorithm; it also couples a mask window to preserve target topology, which enables multi-target localization. Evaluations are conducted on 61 test cases from five images of three node-technologies. The results validate the accuracy, time-efficiency, and the generalizability of the proposed method of locating multi-target from SEM images for hardware assurance applications.