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Rationale: Elastin‐like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products. Methods: ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline‐specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high‐resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed‐phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high‐resolution mass spectrometry using a time‐of‐flight mass spectrometer. Results: Absolute quantification of ELPs was achieved by utilizing ELP‐specific properties, that is, proline‐rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed‐phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N‐terminal modifications. Conclusions: Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs. [ABSTRACT FROM AUTHOR]