Atrial fibrillation (AF) is the most commonly clinically-encountered arrhythmia. Catheter ablation of AF is mainly based on trigger elimination and modification of the AF substrate. Substrate mapping ablation of complex fractionated atrial electrograms (CFAEs) has emerged to be a promising technique. To improve substrate mapping based on CFAE analysis, automatic detection algorithms need to be developed in order to simplify and accelerate the ablation procedures. According to the latest studies, the level of fractionation has been shown to be promisingly well estimated from CFAE measured during radio frequency (RF) ablation of AF. The nature of CFAE is generally nonlinear and nonstationary, so the use of complexity measures is considered to be the appropriate technique for the analysis of AF records. This work proposes the use of sample entropy (SampEn), not only as a way to discern between non-fractionated and fractionated atrial electrograms (A-EGM), but also as a tool for characterizing the degree of A-EGM regularity, which is linked to changes in the AF substrate and to heart tissue damage. The use of SampEn combined with a blin ... mehrd parameter estimation optimization process enables the classification between CFAE and non-CFAE with statistical significance (p < 0:001), 0.89 area under the ROC, 86% specificity and 77% sensitivity over a mixed database of A-EGM combined from two independent CFAE signal databases, recorded during RF ablation of AF in two EU countries (542 signals in total). On the basis of the results obtained in this study, it can be suggested that the use of SampEn is suitable for real-time support during navigation of RF ablation of AF, as only 1.5 seconds of signal segments need to be analyzed.