Background: Antibiotic Associated Diarrhea (AAD) is a common health problem in patients under antibiotic therapy. Clostridium difficile is the commonest cause of AAD. Objectives: our study aimed to compare between cytotoxicity assay and PCR for the diagnosis of Clostridium difficile antibiotic associated diarrhea as regard specificity and sensitivityMethodology: Stool samples were collected from patients clinically suspected to have antibiotic associated diarrhea and proved by sigmoidscope from General Surgery Department in Tanta University. All samples were subjected to cytotoxicity assay to detect C-difficile toxin and PCR to detect ctd B gene encoding for toxin B. Results: Cytotoxicity test showed specificity 96% and sensitivity 90.5%.while PCR assay showed specificity 100% and sensitivity 98%. Conclusion: PCR is rapid, specific and sensitive in the diagnosis of C-difficile Antibiotic associated diarrhea.