We aimed to perform a comprehensive prospective histological analysis in a cohort of 40 inflammatory bowel disease (IBD) patients, both prior to and during biological therapy. The main objective was to establish the impact of histological activity in assessing treatment response and therapy guidance. Biopsy samples were assessed in two different stages of the disease course - before introduction of biological therapy and during follow-up (6-12 months later). Clinical, biochemical and endoscopic parameters were simultaneously gathered. By univariate analysis, presence of lymphoid aggregates, mucin depletion and ulcerations had a strong association with treatment response. IBD diagnosis >5 years and previous biological therapy were the most susceptible to treatment non-response. Nancy Index (NI) >1 is highly accurate, sensitive and specific in defining treatment responders. In addition to NI, high histological burden (mucin depletion, lymphoid aggregates and ulcerations) seems to predict treatment response in biological therapy-treated patients. Instead of a stringent "histological remission" goal, histological improvement of individual parameters might be a more reasonable goal, avoiding the risk of overtreatment. Further research is needed to assess histological burden in patients with endoscopic remission in order to stratify low- vs. high-risk "relapsers" and to avoid under- or over-treatment.