This study evaluated two methods, namely high performance liquid chromatography with fluorescence detection (HPLC-FLD) and Vibrio harveyi BB170 bioassay, for autoinducer-2 (AI-2) quantification in marine samples. Using both methods, the study also investigated the stability of AI-2 in varying pH, temperature and media, as well as quantified the amount of AI-2 signals in marine samples.HPLC-FLD method showed a higher level of reproducibility and precision compared to V. harveyi BB170 bioassay. Alkaline pH > 8 and high temperature (> 37°C) increased the instability of AI-2. The AI-2 concentrations in seawater were low, ca. 3.2-27.6 pmol l-1 whereas 8- week old marine biofilm grew on an 18.8 cm2 substratum accumulated ca. 0.207 nmol of AI-2.Both methods have pros and cons for AI-2 quantification in marine samples. Regardless, both methods reported a ubiquitous presence of AI-2 in both planktonic and biomass fractions of seawater, as well as in marine biofilm.In this study, AI-2 signals were for the first time enumerated in marine samples to reveal the ubiquitous presence of AI-2 in this environment. The findings suggest a possible role of AI-2 in biofilm formation in marine environment, and the contribution of AI-2 in biofilm-associated problems such as biofouling and biocorrosion. This article is protected by copyright. All rights reserved.