Multiplexed detection has been a big motivation in biomarker analysis as it not only saves cost and labor but also improves the reliability of diagnosis. Among the many approaches for multiplexed detection, fluorescence resonance energy transfer (FRET)-based multiplexing is gaining popularity particularly due to its low background and quantitative nature. Although several FRET-based approaches have been developed for multiplexing, they require either multiple FRET pairs in combination with multiple excitation sources or complicated algorithms to accurately assign signals for individual FRET pairs. Therefore, the need for multiple FRET pairs and multiple excitation sources not only complicates the experimental design but also increases the cost and labor. In this regard, multiplexed sensing by tuning the interdye distance of a single FRET pair could be an ideal solution if identification of multiple FRET efficiencies in a single imaging is possible. Here, implementing a program called MASH-FRET, we evaluated the rigor and capability of this program in identifying seemingly overlapped FRET populations obtained from a multiplexed detection experiment using a single FRET pair. Through MASH-FRET-enabled bootstrap-based analysis of FRET data (also called BOBA-FRET), we demonstrated that the resolution and statistical confidence of the poorly resolved or even unresolved FRET populations can be readily determined. Using simulated FRET data, we further demonstrated that the program can easily identify FRET populations separated by ∼0.1 in mean FRET values, indicating an upper limit of ∼9-fold multiplexing without the need for complicated labeling schemes and multiexcitation sources. Therefore, this paper presents a data analysis approach on an existing platform that has a great potential to simplify the technological needs for multiplexing and to broaden the scope of FRET-based single-molecule analyses.