In the present work, an automatic method based on multisyringe flow injection analysis (MSFIA) was developed for determination of scavenging capacity against H 2 O 2 using a non-enzymatic fluorimetric assay for H 2 O 2 detection based on the formation of europium-tetracycline-H 2 O 2 complex. The MSFIA-fluorimetric methodology was developed to perform in-line the scavenging reaction of H 2 O 2 prior to reaction of the remaining H 2 O 2 with the fluorescent probe, using conditions close to those found in vivo regarding pH (6.9), temperature (37°C) and H 2 O 2 concentration (25μM). This approach allowed the evaluation of scavenging capacity against H 2 O 2 in a non-competitive (antioxidant+H 2 O 2 ) or a competitive (antioxidant+H 2 O 2 +biological target) scheme. Using the first strategy, IC 50 values determined for the antioxidant compounds glutathione reduced (1191±46μM) and pyruvate (446±49μM) were lower than those obtained for biological targets such as cysteine (2616±182μM), taurine (359±38mM) and adenine (2224±214μM), indicating that reactivity towards H 2 O 2 was higher for antioxidant compounds than for biological targets. However, when a competitive scheme was applied, the scavenging effectiveness against H 2 O 2 depended on the biological molecule present, showing that antioxidant assessment should also take into consideration the concomitant reactivity of biological molecules or structures that are prone to oxidative damage.