The growing interest in RNA biology, along with an expansion in computational capabilities, throughput, and sensitivity has provided researchers new tools to probe RNA. RNA selective 2′-hydroxy acylation analyzed by primer extension (SHAPE) utilizes the unique reactivity of the 2′-OH on the ribose sugar at sites of flexibility within an RNA structure to probe the secondary structures of RNA. This work sought to address the limitations of current SHAPE probes including poor aqueous solubility. Several iterations of designs were tested, and these results were used to direct the continued development of new probes. A strategy for modulation of probe reactivity was developed and utilized for isatoic anhydride-based probes and nicotinic acid imidazolide-based probes. The work demonstrated the capability of new RNA probes to provide improved signal to noise of RNA SHAPE both in vivo and in vitro.