A previous study stated that reading holy books can make a meaningful change in heart rate variability (HRV). The purpose of this study was to test the effect of reading the Quran, the heavenly religious book of the Muslim people in the Arabic language, on the Farsi (Persian)-speaking Muslims with various levels of spiritual well-being (SWB). In addition, novel to this study was the assessment of whether or not it is possible to use HRV features to distinguish individuals with high SWB from those with medium SWB. First, a questionnaire was completed by 31 volunteers to measure their SWB. Baseline ECG measurements were recorded during the resting stage. The volunteers were then asked to read the Quran for 5 min while ECG was recorded again. HRV indexes were calculated and four features were extracted and analyzed based on their correlation with the different levels of SWB. Independent t-tests were conducted and the results established a significant difference in these four features between high SWB and medium SWB groups, during the reading stage. Subsequently, with the use of these four HRV features, an artificial neural network and a decision tree were designed to classify the levels of SWB in volunteers. The outcome of this study demonstrated that it is possible to evaluate the level of SWB in individuals while they are reading the Quran.