Madhuca longifolia, Cassia tora, Cocculus hirsutus, Celosea argentea and Alternanthera sessilis plants were scrutinized to determine the amino acid content by a validated method. Herein we report a novel, accurate and cost-effective reversed-phase high-performance liquid chromatography method for the simultaneous determination and quantification of 14 amino acids. The developed method was validated as per the International Conference on Harmonisation guidelines. All calibration curves showed a good linear relationship (r > 0.9928) within test range. Precision was assessed by intra-and inter-day tests with relative standard deviations less than 2%. Recovery of 98.38-99.60% with relative standard deviations < 1.00% was observed. On quantification, glutamic acid, aspartic acid, alanine, methionine, glycine, leucine, valine and threonine were present in major quantity from different parts of selected plants which is advantageous as they control most of the physiological activities of the body. The proposed method can be used for the simultaneous estimation of 14 amino acids in one run and can be extended for amino acid-based standardization of herbal formulations containing these plants and its use in pharmaceutical industries.