Folin Ciocalteu (F-C) assay is the most widely used and convenient method to determine the total phenolics content in foods, herbs, and other plant extracts. Different phenolics standards such as gallic acid, ferulic acid, chlorogenic acid, catechol, and vanillic acid have been used for calibration curves in this assay method. Comparison of these standards, in single or combination of two or more, for more accurate determination of phenolics has not been reported so far. This study tested five single phenolics and seven combinations of mixed phenolic standards to evaluate the optimal standards for F-C method. The different standards were tested to calculate the phenolic content in three known test solutions. We also evaluated interference effect of various compounds in phenolics estimation by F-C method that is usually present in the lignocellulosic biomass-derived sugar solution, and in food products along with phenolics. Finally, the optimal standards with five phenolics were used for the determination of phenolics in alkali pretreated biomass extract. The results indicated that gallic acid was the best standard among the single phenolic compounds and five phenolic compounds solution was the best standard among the mixed phenolic solutions. The presence of glucose, HMF, furfural, and vitamin-B12 did not interfere in phenolic determination; whereas ascorbic acid, tyrosine, formic acid, and acetic acid strongly interfered the results. The results also showed that biomass pelleting process did not affect the generation of phenolics in alkali pretreatment extract.