BACKGROUNDSpinach is well recognized as a functional food owing to its diverse nutritional composition, including polyphenols. Freezing is an efficient preservation method that is used to maintain the physical and nutritional characteristics and extend the shelf life of spinach. The aim of this study was to determine the changes in polyphenols in the samples taken from various production steps of the industrial freezing process of spinach, and to evaluate the bioaccessibility of these bioactive compounds for raw material, by‐product, and frozen product using the standardized in vitro digestion model simulating the digestion in the mouth, stomach, and intestine.RESULTSUltra‐performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and high‐performance liquid chromatography equipped with photodiode array detection analysis of spinach samples led to the identification of eight flavonoids and two phenolic acids. The changes occurring in flavonoids after blanching, chopping, and freezing steps were statistically not significant compared with the raw material (P > 0.05). On the other hand, by‐product was found to contain significantly lower amounts of flavonoids (98% in total) and phenolic acids (90% in total) (P < 0.05) compared with the raw material. Furthermore, after in vitro digestion, frozen spinach was found to contain higher amounts of bioaccessible flavonoids (15% in total) and phenolic acids (16% in total) compared with fresh spinach.CONCLUSIONOverall, the current study highlighted that industrial freezing might be a good strategy to preserve the polyphenol content of fresh spinach as well as to enhance the total amount of bioaccessible polyphenols.