Identification of the causative organism(s) in periprosthetic joint infection (PJI) is a challenging task. The shortcomings of traditional cultures have been emphasized in recent literature, culminating in a clinical entity known as “culture-negative PJI.” Amidst the growing burden of biofilm infections that are inherently difficult to culture, the field of clinical microbiology has seen a paradigm shift from culture-based to molecular-based methods. These novel techniques hold much promise in the demystification of culture-negative PJI and revolutionization of the microbiology laboratory. This article outlines the clinical implications of culture-negative PJI, common causes of this diagnostic conundrum, established strategies to improve culture yield, and newer molecular techniques to detect infectious organisms.