The purpose of this study is to investigate the role of serum calprotectin (CP), lactoferrin (LF), and high-mobility group protein B1 (HMGB-1) levels and fecal CP and LF levels in differential diagnosis of acute uncomplicated appendicitis from other causes of abdominal pain and further from complicated appendicitis. Totally, 120 children were included grouped into 4 as: healthy controls, patients with right lower quadrant pain with other than surgical causes, patients with uncomplicated appendicitis, and patients with complicated appendicitis. Serum CP, LF, HMGB-1, C-reactive protein (CRP) levels, and white blood cell (WBC) count were studied as well as the fecal CP and LF levels. There was a statistically significant difference between control group and both uncomplicated and complicated acute appendicitis groups, regarding all parameters. In diagnosis of complicated acute appendicitis, area under curve (AUC) for fecal LF, serum CP, and serum HMGB-1 were determined as 1.00 and the cutoff level was determined as 25 μg/g feces, 670 ng/mL, and 30 ng/mL, respectively. In differential diagnosis of uncomplicated and complicated AA, the most accurate parameter was fecal LF with an AUC of 0.977. At a 60 μg/g cutoff value for this variable, sensitivity, specificity, and accuracy were 96.7, 93.3, and 95.0 %, respectively. In conclusion, HMGB-1, calprotectin, and lactoferrin constitute novel markers in diagnosis of AA. Moreover, their levels may be helpful for the clinicians to judge about the severity of the condition. Larger studies are warranted to determine the diagnostic potential of HMGB-1, LF, and CP in AA diagnosis.