AbstractObjectivesUrine cytology is the most widely used noninvasive screening tool for urothelial carcinoma diagnosis and surveillance. Although highly specific, urine cytology exhibits suboptimal sensitivity. This study aimed to determine whether hTERT immunocytochemistry (ICC) could be applicable as an ancillary test in routine cytology practice.MethodsA total of 561 urinary tract samples were initially screened in this study. All of them were prepared using SurePath liquid‐based cytology (LBC), while additional LBC slides were made and subsequently used for hTERT (SCD‐A7) ICC.ResultsFrom the 561 samples screened, 337 were finally analyzed, all having an adequate cellularity and available follow‐up histology. The hTERT ICC‐positive rate was 95.9% (n= 208/217), 96% (n= 24/25), and 100% (n= 4/4) in cytology samples with high‐grade urothelial carcinoma, carcinoma in situ, and low‐grade urothelial carcinoma subsequent histology. Among the 64 atypical cytology cases histologically confirmed as urothelial carcinomas, 92.2% (n= 59/64) were immunoreactive to hTERT, whereas the two histologically benign cases were ICC‐negative. 87/90 (96.7%) of the cytology cases confirmed to be benign in follow‐up were hTERT‐negative. The overall sensitivity and specificity of hTERT ICC were 96.3% and 98.8%, respectively (AUROC = 0.963; 95% CI = 0.960–0.967).ConclusionsThe hTERT ICC test exhibited consistent and intense staining in malignant urothelial cells, suggesting its value as an ancillary test in liquid‐based urine cytology.