The current study was undertaken to compare four analytical methods including drop collapse, oil spreading, surface tension (SFT) measurements, and blood agar lysis tests for detection of biosurfactant-producing bacteria. Among 32 biosurfactant-producing bacteria isolated from Ahvaz oil fields, in south of Iran, 16 isolates (50%) exhibited highest biosurfactant production. Eleven isolates (MASH.1 to MASH.11) demonstrated a reduction in surface tension from 65 mN/m to less than 41 mN/m. The results showed that about 91% of these highly biosurfactant producers had the same response levels of "++++" and "+++" in the case of both SFT and oil spreading methods. Among these, seven isolates had the haemolysis diameter less than 1 cm or between 1 and 2 cm on blood agar. As 64% of the best biosurfactant producers did not completely lyses blood, the ability of biosurfactant-producers for haemolysis may not always be trustworthy. According to our data, there is a good consistency between oil spreading technique and surface tension. As a conclusion, oil spreading method is the fastest, simplest and most consistent analytical method to be suggested for accurate measurements of biosurfactant producers.