Abstract The study aims to determine the phytochemical constituents present in the cell suspensions of cotton elicited by MeJA and ethephon. Elicitors were added alone or in combination. The freeze-dried cells were soaked with 10 mL of methanol 96% and purified through a mini-column of C18. After concentration of filtrate, the dry extract was dissolved in methanol with chromatographic grade and filtered through a Millipore membrane with 0.22 μm porosity. Then, each of the extracts was further subjected to Gas Chromatography-Mass Spectrometry (GC-MS). Results showed six compounds in control, seven in MeJA, eight in ethephon and nine in co-treatment MeJA/Ethephon. In total, 22 different compounds were detected in the cell suspensions by elicitor or not. Thirteen of these compounds were identified with three in control, four in Ethephon, six in MeJA and four in MeJA/Ethephon co-treatment. Most of identified compounds are lipids and involved in endogenous MeJA metabolism, including 3–oxo–2-(2‘-pentenyl)-cyclopentane-1-octanoic acid by the octanoic pathway. This compound predominates with a rate of 100% under MeJA/Ethephon treatment and 75% under MeJA alone treatment. The findings suggest that Nitro-tert-butyl-acetate, 4-O-α-D-glucopyranosyl-D-glucose, Octanoic acid ethyl ester and Monolinoleoylglycerol trimethylsilyl ether under Ethephon treatment, n-decanoic acid, hexanoic acid, D-glycero-D-ido-Heptose, 6-Hepatadecyl-5,6-dihydro-2H-pyran-2-one and Bicyclo [3.3.1] nonan-9-one 1,2,4-trimethyl-3-nitrile under MeJA treatment and, 4,5-diethyl-3,6-dimethyl-3,5-octadiene under concomitant application of MeJA and Ethephon may be potential biomarkers of the response to cotton cell suspensions elicitation by MeJA and ethephon. This study demonstrates that elicitors application can affect highly the biosynthesis of compounds in cotton using cell suspensions as model.