The measurement of airborne endotoxins is thus far not standardized. Earlier studies reported higher endotoxin yields when Tween 20 was added to the media used for filter extraction and in the Limulus amebocyte lysate (LAL) assay. This study compared four common media and assessed the effects of Tween during extraction and analysis separately. Parallel airborne dust samples from five work environments (n = 250) were used to compare the four media (pyrogen-free water [PFW], PFW-Tween 20, PFW-Tris, and PFW-triethylamine-phosphate [TAP]) and an extraction time of 10 or 60 min. A subset of the extracts in PFW or PFW-Tween (n = 40) were analyzed in parallel LAL assays with PFW or PFW-Tween as the assay medium. The results produced by a shorter extraction time or the presence of Tris were similar to the results for the reference procedure (PFW and 60 min of shaking). The use of PFW-TAP showed overall lower yields and a deviant calibration curve. The presence of Tween in the extraction medium resulted in significantly (P < 0.05) higher endotoxin yields from all dust types, independent of the effect of Tween in the assay. Tween in the LAL assay, however, also strongly inhibited the reactivity of the lipopolysaccharide (LPS) standard, thus shifting the calibration curve to higher values. The inhibition of LPS in test samples was less pronounced and varied between dust sources, resulting in enhanced calculated concentrations. This assay effect could be circumvented by diluting extracts at least 50-fold before the LAL assay. In conclusion, of the media tested, only Tween enhances the efficiency of endotoxin extraction from airborne dust samples in a consistent manner. We recommend extraction in PFW-Tween combined with dilution and LAL analysis in PFW.