Ryanodine receptors (RyRs) are large-conductance Ca²⁺ channels located at the membrane of endoplasmic reticulum (ER) or the muscle equivalent, sarcoplasmic reticulum (SR). Three isoforms of mammalian RyRs are expressed across tissues and regulate diverse cellular physiology. Although various chemicals have been identified as agonists and antagonists of RyRs, the isoform-selective pharmacological manipulation is still challenging. In this study, we screened selective agonists of type 1 Ryanodine receptor (RyR1) and investigated the structure-activity relationships. The ortho/meta/para-orientation and the length of alkyl chains affect their potency. Furthermore, experiments with RyR1 mutants revealed the amino acid residues responsible for the channel activation by agonists. The novel agonists identified in this study are prospective tools for the isoform-selective pharmacology of RyRs.