AbstractPyrolysis processes are an alternative to minimize the environmental problem associated to agrifood industrial wastes. The main product resulting from these processes is a high‐value liquid product, called bio‐oil. Recently, the use of comprehensive two‐dimensional liquid chromatography (LC × LC) has been demonstrated as a useful tool to improve the characterization of the water‐soluble phases of bio‐oils, considering their complexity and high water content. However, the precise composition of bio‐oils from different agrifood byproducts is still unknown. In the present study, the qualitative and quantitative screening of eight aqueous phases from different biomasses, not yet reported in the literature, using LC × LC is presented. The two‐dimensional approach was based on the use of two reverse phase separations. An amide column in the first dimension together with a C18 column in the second dimension were employed. Thanks to the use of diode array and mass spectrometry detection, 28 compounds were identified and quantified in the aqueous phase samples with good figures of merit. Samples showed a distinct quali‐quantitative composition and a great predominance of compounds belonging to aldehydes, ketones and phenols, most of them with high polarity.