Photoinduced nitric oxide (NO) release for complexes [RuNO(L)2(NO2)2OH], where L = methyl isonicotinate (1), ethyl isonicotinate (2), methyl nicotinate (3) and ethyl nicotinate (4) was studied in DMSO, MeCN and water solutions (PBS, CTAB) using spectroscopic methods (UV‐vis, IR, EPR) and spectrometric techniques (ESI‐МS). Additionally, we present methodological evidence for improving the calculation of the quantum yield (QY) of NO release through the utilization of a combined IR‐UV‐vis‐spectroscopy flow‐through setup. According to DFT calculations, the production of nitric oxide occurs through the photoinduced cleavage of the Ru‐NO bond, triggered by irradiation at 445 or 532 nm. This cleavage is a consequence of charge transfer from the orbitals of the Ru‐OH group and the equatorial ligands (HOMO, HOMO‐1) to the Ru‐NO antibonding orbital (LUMO). The cytotoxicity and photoinduced cytotoxicity of the investigated compounds were assessed against the breast adenocarcinoma cell line MCF‐7. Moreover, the investigation of the lipophilic properties of compounds 1‐4 unveiled a significant influence of their lipophilicity on cytotoxic behavior, allowing for the modification of cytotoxicity through changes in the ligand L or by light irradiation.