Hand eczema (HE) is a prevalent skin disease. However, the classification of HE into different subtypes remains challenging. A limited number of previous studies have employed invasive biopsy–based strategies; yet, studies of the HE proteome using noninvasive tape-stripping methodology have not been reported. In this study, we wanted to assess whether global proteomic analysis of skin tape strip samples can be used for subclassification of patients with HE. Tape strips were collected from patients with HE and healthy skin. Liquid chromatography–mass spectrometry proteomics was performed, and the global protein expression was analyzed. We identified 2,919 proteins in stratum corneum–derived skin cells from tape strip samples. Compared with healthy skin, the lesional samples from patients with HE exhibited increased expression of immune-related markers and a decreased expression of structural barrier proteins. The difference between HE subtypes was restricted to the lesional skin areas and included an increased expression of skin barrier–related proteins independently of the concurrent AD. In conclusion, we found that the noninvasive tape strip method used in combination with liquid chromatography–mass spectrometry proteomics can be used for analysis of skin protein expression in patients with HE. Thus, the method shows potential for assessing the proteomic differences between subtypes of HE and biomarker discovery.