The presence of non-specific amplification (NSA) in bioanalytical RCA assays could lead to false-positive readouts, and background signals, and would adversely affect analytical sensitivity. Despite significant advances in RCA-based methods, the possible origin of NSA in RCA or the means to mitigate it remains unexplored. Herein, we assessed the role of ligation and subsequent exonuclease digestion steps in non-specific rolling circle amplification for presynthesized circular DNA (cDNA) substrates. Three different ligation techniques, namely selfannealed, cohesive end, and splint-padlock mediated ligation were utilized to prepare cDNA candidates that were then subjected to nine different digestion conditions spanning a combination of enzymes, buffers, and salt conditions. NSA was measured for both linear RCA and branched amplification with an HRCA primer on the cDNA, and the resulting fluorescence from SYBR Green I intercalation was quantified in the rt-PCR instrument. In general, the dual Exo I and III digestion in presence of Exo III buffer alone was the most effective in reducing NSA, even more so than a 1:1 mix of Exo I and III buffer. Besides, more efficient digestion was observed for some of the substrates when a higher concentration of Mg 2+ (up to 33 mM) was present.