AbstractUse of solid phase microextraction (SPME) for cell culture metabolomic analysis allows for the attainment of more sophisticated data from in vitro cell cultures. Moreover, considering that SPME allows the implementation of multiple extractions from the same sample due to its non/low-depletive nature, time course studies using the same set of samples are thus facilitated via this method. Such an approach results in a reduction in the number of samples needed for analysis thus eliminates inter-batch variability related to biological variation occurring during cell culturing. The current work aims to demonstrate the capability of SPME for measurements of combretastatin A4 (CA4) effectiveness on non-small cell cancer cell line. A cultivation protocol was established in the 96-well plate, and a fiber format of SPME was selected for metabolite extraction. The extracellular metabolic pattern of cells was changed after administration of the tested drug. This suggests pharmacological activity of the administered compound towards the studied cell line model. Results support that the use of direct immersion SPME for analysis of cell cultures does not affect cells growth or contaminate sample. Consequently, SPME allows the attainment of accurate information regarding drug uptake, metabolism, and metabolomic changes in the studied cells induced by exposure to the drug simultaneously in a single experiment.