Objective To assess the feasibility of autofluorescence spectroscopy in the diagnosis of cervical intraepithelial neoplasia (CIN) using broadband light excitation. Design Feasibility study. Setting Colposcopy clinic of an university hospital. Population Sixty‐eight patients at risk for CIN. Methods After excitation with a broadband light between 375 and 440 nm, spectral distribution of native tissue fluorescence (autofluorescence) was acquired from 685 cervical sites for the localisation and differentiation of CIN, and compared with colposcopically directed biopsy and human papillomavirus (HPV) DNA testing. Main outcome measure Detection of CIN. Results The evaluation of spectral measurements revealed significantly lower autofluorescence values for CIN 3 lesions compared with normal tissue (P < 0.001), and compared with CIN 1 or CIN 2 (P < 0.002). High grade CIN lesions (CIN 2/3) presented with a significant reduced autofluorescence compared with CIN 1 (P < 0.002). Patients with a positive HPV DNA testing showed a significantly lower autofluorescence than patients tested negative for HPV DNA (P < 0.05). Severe inflammation such as chronic cervicitis may lead to false positive results. Conclusions Autofluorescence spectroscopy represents an interesting approach for the detection of cervical neoplasia. Using an excitation wavelength band between 375 and 440 nm, significant differences between normal and precancerous lesions of the cervix can be seen.