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In this paper, we present the concept of traceback analysis to identify the origins of non-wet defects caused by hydrophobic unknown nanoparticles in the semiconductor packaging process. Traceback analysis involves five-steps, i.e., practical observation of failure mode, detailed physical and chemical analysis, process investigation, hypothesis establishment, and failure reproduction with comparison. Initially, the morphology and composition of the unknown nanoparticles are analyzed using an optical microscope (OM), field-emission scanning electron microscopy (FE-SEM), contact angle (CA), energy-dispersive spectroscopy (EDS), and Fourier-transform infrared spectroscopy (FT-IR). As a result, spherical unknown nanoparticles with a size of ca. 500 nm consists of organic imidazole-derived materials, similar to the composition of organic solderability preservative (OSP). To identify the origin, printed circuit board (PCB) manufacturing processes are reviewed step-by-step. Traceback analysis identifies that the 1-methoxy-2-propyl acetate (PGMEA) cleaning solvent can form spherical emulsions when mixed with deionized (DI) water during the cleaning process. This can occur when PGMEA and DI water mix in the cleaning bath or recycling pipes, or when leftover PGMEA and DI water mix during next cleaning processes. These PGMEA/DI water form emulsions owing to the polarity differences between hydrophobic PGMEA and hydrophilic DI water and remain on the PCB surface. Thus, organic nanoparticles grow in the PGMEA emulsion on the Cu pad during OSP application for surface finishing purposes. This comprehensive approach ensures a thorough understanding of defect origins. The traceback analysis described in this study, involving five-steps, is applicable not only in semiconductor packaging but also in defect investigation across various industries.