Background and aims As mucosal healing is the goal of treatment in inflammatory bowel disease, defining a faecal (f-) calprotectin cut-off level for mucosal healing is crucial. Previous studies have presented different cut-off levels. The aim of this study was to investigate the ability of two f-calprotectin assays to differentiate mucosal healing from inflammation in ulcerative colitis. Methods Sixty-two patients with ulcerative colitis underwent colonoscopy for classification of mucosal inflammation (Mayo endoscopic subscore). The patients also submitted a faecal sample for f-calprotectin analysis using two different ELISA assays, Calpro ELISA and Buhlmann ELISA. Results The two assays correlated significantly, with a Spearman rank correlation coefficient of 0.86. Both assays showed significantly different f-calprotectin levels in patients with a Mayo endoscopic subscore of 0 (mucosal healing) and 1-3 (inflamed mucosa) (p < 0.001). Using ROC curve analyses we selected the best cut-off levels for both assays with responding sensitivity and specificity (presented with 95% confidence intervals); Calpro ELISA cut-off 61 μg/g, sensitivity 84.1 % (75.0-93.2%), specificity 83.3 % (74.0-92.6%), and Buhlmann ELISA cut-off 96 μg/g, sensitivity 90.9 % (83.7-98.1%), specificity 83.3 % (74.0-92.6%). Defining mucosal healing as a Mayo endoscopic subscore ≤ 1, cut-off levels increased; Calpro ELISA cut-off 110 μg/g, sensitivity 80.0 % (70-90%), specificity 66.6 % (54.9-78.3%), and Buhlmann ELISA cut-off 259 µg/g, sensitivity 83.3 % (74-92.6%), specificity 71.9 % (60.7-83.1%). Conclusions The study demonstrates the need for assay specific cut-off levels in clinical practice, as the f-calprotectin cut-off level for endoscopic disease activity differed in these two assays.