The overproduction of free radicals and reactive oxygen species (ROS) has been proved as a basic damage mechanism and cause for oxidative stress. Their measurement is often hindered by the low signal. This could be resolved with the application of luminescent probes (lophines, luminol, lucigenin, etc.). The focus of this study is to synthesize and describe the spectral properties and physicochemical characteristics of lophine and its derivatives as new chemiluminescent in vitro activators. The prepared luminophores are analogues of lophine. Their absorption maxima are in the range 329-340 nm, with good-to-high extinction coefficients. Their spectral properties are measured in methanol and buffer solutions with pH 3.5, 7.4 and 8.5. Same conditions were applied in the systems for chemiluminescent assay in vitro: (1) Fenton's (Fe(2+)+H2O2) for the generation of ·OH and -OH species, (2) Hydrogen peroxide (H2O2), (3) Iron (II) sulfate (FeSO4), (4) Glutathione-peroxidase, monitoring the deactivation of H2O2, (5) Ascorbic acid-Fenton's reagent: Vit.C appears a strong oxidant, generating free-radical products when applied in higher than physiological concentrations, (6) Reduced α-nicotinamide adenine dinucleotide (NADH)-phenazine methosulfate-for the generation of superoxide radicals (O2 (·-)). Lophine and all novel compounds do not alter the kinetics, except of the dimethyl amino substituted derivative (4-(3a,11b-dihydro-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)-N,N-dimethylaniline) in the glutathione-peroxidase system, at pH 8.5. Same derivative showed a comparable or higher activity than Lucigenin and Rhodamine 6G. In neutral and acidic medium, in the Fenton's system, Rhodamine 6G was the most appropriate probe. In alkaline pH and oxidant H2O2, Lucigenin induced a signal twice as strong as the signal compared to all other activators.