Single-molecule: FRET is One Of the most powerful and widely used biophysical techniques in biological sciences. It, however often suffers from limitations such as weak signal and limited measurement time intrinsic to single- Molecule fluorescence measurements. Despite several ameliorative measures taken to increase- measurement time, it is nearly impossible to acquire meaningful kinetic information on a molecule if conformational transitions of the molecule are ultraslow such that transition times ((orig)) are. comparable to or longer than measurement times (delta t) limited by the finite lifetime of fluorescent dye. Here, to extract a reliable and accurate mean transition time from, a series of short time traces with ultraslow kinetics, we suggest seheme called sHaRPer (serialized Handshaking Repeated Permutation with end removal) that concatenates multiple time traces because data acquisition frequency f and measurement time (delta t) affect the estimation of mean transition time (), we provide Mathematical criteria that f, delta t, and should satisfy to make close enough to (orig) Although application of the sHaRPer: methods a potential risk of distorting the time,constants of individual kinetic phases if the data are described with kinetic partitioning, We, also provide criteria to avoid such distortion. Our sHaRPer method is a useful way to handle single-molecule data:With,slow transition kinetics This,study:provides a practical vide to use sHaRPer.