A high-performance thin-layer chromatography (HPTLC)-based sensitive, rapid and stringent protocol is designed for detection and quantification of five phytohormones simultaneously. Culture filtrate of Pseudomonas bacteria was acidified with 7 M HCl and extracted with an equal volume of ethyl acetate to separate abscisic acid (ABA), jasmonic acid (JA), gibberellic acid (GA(3)), and indole-3-acetic acid (IAA). Kinetin was extracted from the remaining water fraction of the same extract. Various extracts were loaded on silica gel 60 F-254 foil using Linomat 5 spray on applicator. Standard phytohormones were also loaded adjacent to the sample, and the foils were developed with isopropanol-ammonia-water [10:1:1 (v/v)] as the mobile phase. A quantitative estimation of the separated ABA, kinetin, JA, GA(3), and IAA was performed by measuring the absorbance at 260, 275, 295, 265, and 280 nm, respectively. HPTLC method was found to be cost effective, robust technique that can be routinely used for simultaneous phytohormone detection in plant or bacterial samples. The present work is not only useful for detection and quantification of phytohormones but also for screening of phytohormone producing microorganisms.