INTRODUCTION: The use of liquid-based cytology (LBC) in non-gynecological fields has progressively increased. However, studies focusing on the application of LBC in bile cytology are limited, and its efficacy remains uncertain. In this study, we assessed the potential of LBC in bile cytology by evaluating the interobserver agreement. METHODS: Bile cytology specimens were collected between 2015 and 2022 by using endoscopic retrograde cholangiopancreatography. Eleven cytotechnologists participated in the evaluation. Digital images of bile cytology specimens prepared using conventional smear (CS), ThinPrep LBC (TP-LBC), and SurePath LBC (SP-LBC) methods (20 cases per preparation method) were assessed for interobserver agreement on 21 diagnostic criteria. The diagnostic accuracy was evaluated using 50 bile cytology cases per preparation method. RESULTS: High interobserver agreement (exact kappa >
0.6) was observed for features, including the loss of nuclear polarity, irregular nuclear spacing, nuclear size variation, and increased nuclear-to-cytoplasmic ratio across the CS, TP-LBC, and SP-LBC methods. Malignant cells in TP-LBC appeared smaller, rounder, and formed flat aggregates compared with those in CS. In contrast, the malignant cells in SP-LBC formed three-dimensional clusters that overlapped and exhibited higher cellularity than those in CS. The sensitivity of bile cytology was 76.4%, 84.8%, and 93.2% for CS, TP-LBC, and SP-LBC, respectively. CONCLUSION: The observers consistently recognized malignant cell features in bile cytology, irrespective of the preparation method. Understanding common features and method-specific cellular morphology is crucial for enhancing diagnostic accuracy. Our findings suggest that LBC methods can be effectively applied to bile cytology, potentially offering improved diagnostic accuracy compared with conventional methods.