To evaluate the feasibility and utility of intraoperative post-conisation human papillomavirus (IOP-HPV) testing and cytology to detect treatment failure in patients with cervical intraepithelial neoplasia grades2-3 (CIN2-3). Prospective observational pilot study. Barcelona, Spain. A cohort of 132 women treated for CIN2-3 by loop electrosurgical conisation. An endocervical sample was obtained intraoperatively with a cytobrush from the cervix remaining after the conisation. The material was kept in PreservCyt medium and processed for Hybrid Capture2 and cytology. Patients were followed-up for 24months. The performance of IOP-HPV testing and IOP cytology was compared with conventional indicators of recurrence (cone margin, endocervical curettage, and HPV testing and cytology at 6months). Treatment failure (i.e. recurrent CIN2-3 during follow-up). Treatment failure was identified in 12 women (9.1%). IOP-HPV testing for sensitivity, specificity, and positive and negative predictive values for treatment failure were 91.7, 78.3, 62.2, and 96.0%, respectively, which are similar to the figures for conventional HPV testing at 6months (91.7, 76.0, 64.0, and 95.1%, respectively), and are better than the values of other conventional predictive factors (cone margin, endocervical curettage, and cytology intraoperative at 6months). IOP-HPV was strongly associated with treatment failure in the multivariate analysis (OR 15.40, 95%CI 1.58-150.42). IOP-HPV testing is feasible, and accurately predicts treatment failure in patients with CIN2-3. This new approach may allow an early identification of patients with treatment failure, thereby facilitating the scheduling of an attenuated follow-up for negative patients who are at very low risk of persistent disease.