Pseudomonas aeruginosa is one of the most important pathogenic bacteria related to biofilm infections. Due to the biofilm multi-drug resistance, methods of biofilm formation enumeration are of interest for assessment of efficient drug regimen development for biofilm inhibition or eradication. There are many different assay methods to determine the biofilm formation, using vital or non-vital dyes. The primary aim of the current study was to develop an assay using a member of tetrazolium salts family, 2,3,5-triphenyl-tetrazolium chloride (TTC), for detection of P. aeruginosa biofilm formation in 96-well microtiter plates and also a method of Minimum Biofilm Inhibitory Concentration (MBIC) determination of antibiotics against P. aeruginosa PAO1. Furthermore, the assay was optimized for TTC concentration, wavelength and period of incubation for 4 different antibiotics. The optimized condition was then compared with two other prevalent methods: the crystal violet (CV) assay and the 2,3-bis (2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. In general, the optimized TTC assay (0.5% TTC, 6h of incubation and absorbance measurement at 405nm for biofilm assay and 1% TTC, 5h of incubation and absorbance measurement at 490nm for MBIC determination) distinguished between biofilms formed by different concentrations of bacteria and also was able to detect lower amounts of biofilm formed in contrast to the other two assay methods suggesting that TTC assay is more sensitive and also less expensive than other vital staining methods.