Background & objectives: Data regarding tumor marker usefulness in diagnosing ascites of unknown etiology and determining its malignant nature are conflicting. We aim to assess the diagnostic value of ascitic and serum tumor markers in ascites of unknown etiology and to evaluate their usefulness besides other laboratory tests in a diagnostic work-up in those patients. Design & setting: A prospective case-control study conducted at Assiut University hospital and oncology institute. Patients & Methods: Three groups were included; Group I: 41 patients with ascites of unknown etiology Group II: 7 Patients with TB ascites and Group III:14 patients with cirrhotic ascites. We assessed the CEA, CEA mRNA, CA15-3, CA19.9, CA125, AFP and PSA in serum and ascetic fluid. A diagnostic work-up for group I included: IPD test, ultrasound, CT, ascetic fluid cytology, SAAG, Laparotomy and biopsy. Results: Ascetic fluid and serum levels of CA15-3 and CA125 were significantly increased in group I and were significantly increased in histopathologically proved malignant ascites compared to TB and cirrhotic ascites. In group I, CA 125 was significantly higher in ascites than serum. With the exception of PSA, all tumor markers significantly correlated in serum and ascetic fluid. No significant difference in the level of ascetic CEA messenger RNA was detected between the 3 groups. Cytology had 53% sensitivity, 94% specificity and CA 125 & CA15-3 had 81% sensitivity and 75% specificity in detection of malignant ascites repectively. Laparotomy and biopsy: diagnosed malignnancy in 53.3% and TB in 13.3%. Conclusions: A diagnostic work-up including SAAG, tumor markers in the serum and ascetic fluid may help in adjunct with ascetic fluid cytology, laparotomy and biopsy, imag- ing and other laboratory tests in diagnosing ascites of unknown etiology