<p>This study presents an optimized protocol for measuring singlet oxygen (¹O₂) quantum yield using a two-component chemical trap consisting of imidazole (ImH) and 4-nitroso-N,N-dimethylaniline (RNO) for water-soluble phthalocyanines (Pcs). Deprotonation of nitrogen atoms in a cavity of metal-free Pc upon addition of ImH to aqueous solution, as well as coordination of ImH to the metal center in a Pc cavity causes changes in the spectral and photophysical properties of a Pc, which could potentially compromise the accuracy of ¹O₂ yield determination. Through a systematic investigation, we examined the influence of ImH concentration, solution pH, and ionic strength (adjusted using different salts) on the ImH-Pc interaction. The optimal measurement conditions were determined to be a 0.5 mM ImH concentration in neutral pH buffer solution with ionic strength exceeding 100 mM. These specific parameters were found to minimize ImH-induced alterations in Pc spectral characteristics while maintaining reliable ¹O₂ yield quantification. The validity of this approach was confirmed through comparative measurements in phosphate buffered saline (PBS) using both the ImH-RNO system and a reference cationic anthracene trap TMAPA, which showed consistent results. These results demonstrate the feasibility of using an optimized protocol to evaluate the photodynamic activity of different Pcs using the ImH-RNO system.</p>