Though audio source separation offers a wide range of applications in audio enhancement and post-production, its performance has yet to reach the satisfactory especially for single-channel mixtures with limited training data. In this paper we present a novel interactive source separation framework that allows end-users to provide feedback at each separation step so as to gradually improve the result. For this purpose, a prototype graphical user interface (GUI) is developed to help users annotating time-frequency regions where a source can be labeled as either active, inactive, or well-separated within the displayed spectrogram. This user feedback information, which is partially new with respect to the state-of-the-art annotations, is then taken into account in a proposed uncertainty-based learning algorithm to constraint the source estimates in next separation step. The considered framework is based on non-negative matrix factorization and is shown to be effective even without using any isolated training data.Index Terms-Interactive audio source separation, nonnegative matrix factorization, uncertainty-based learning, timefrequency annotation, user feedback.