Spontaneous bacterial peritonitis (SBP) is considered the paradigmatic model of infection in patients with liver cirrhosis. Therefore, there is a need for an accurate and rapid method for SBP diagnosis. The aim of this study was to evaluate the validity of serum-ascites 25-hydroxyvitamin D (25-OH vitamin D) gradient (SADG) as a marker for diagnosing SBP in patients with cirrhotic ascites. We conducted a cross-sectional analytic study of 88 patients with portal hypertensive ascites resulting from liver cirrhosis of any etiology. The demographic, clinical, and laboratory characteristics of the patients were recorded. The level of 25-OH vitamin D in serum and ascitic fluid was measured using high-performance liquid chromatography autoanalyzer. The SADG was calculated with the formula: 25-OH vitamin D in serum - 25-OH vitamin D in ascites. Vitamin D deficiency was detected in 89.8% of the studied patients. The SADG values ranged between 0 and 69.2 ng/mL, with a median value of 5.58 ng/mL. It was significantly lower in patients with SBP than in those without SBP (P = .004). The area under the curve for SADG in exclusion of SBP was 0.67 at a cutoff value of ≥5.57 ng/mL. We found that SADG may be a valid marker of SBP in patients with cirrhotic ascites.