A number of bioactive brominated secondary metabolites, including hydroxylated polybrominated diphenyl ethers, have been isolated from algae, sponges, and bacteria. In the present study, a screening method using liquid-chromatography tandem mass spectrometry was developed for the identification and selective determination of dihydroxy (diOH), hydroxy-methoxy (OH-MeO), and dimethoxy (diMeO) analogs of tetra- to hexa-BDEs in marine biota. In negative atmospheric pressure chemical ionization (APCI) mode, diOH and OH-MeO analogs provided intense [M-H](-) ions, whereas diMeO analogs provided characteristic [M-Br+O](-) and [M-CH(3)](-) ions. This enabled the diOH-, OH-MeO-, and diMeO-PBDEs to be distinguished using selected reaction monitoring transitions in the APCI source. Recoveries of 2'-OH-6-MeO-2,3',4,5'-tetra-BDE in spiked marine samples were 84 ± 5 %, with a limit of quantification at 9.1 ng mL(-1) (signal-to-noise ratio = 10). The developed method was used to analyze two sponge species collected from Palau, Micronesia; the concentration ratio of diOH-tetra-BDE:OH-MeO-tetra-BDE was 10:1 for the Lamellodysidea sp., whereas it was 1:30 for the Callyspongia sp.