Background: Natural moisturizing factor (NMF) is used as genotypic and phenotypic biomarker in diagnostics. This study is a side-to-side comparison of two different methods to determine NMF in atopic dermatitis patients: Raman microspectroscopy and stratum corneum tape stripping followed by HPLC. Results: Measured NMF values were significantly correlated (R2 =.61; p <.0001), both methods demonstrated a concentration-depth dependence of NMF and reduced NMF levels in the carriers of filaggrin null mutations. Good agreement between measurements of left and right arms indicated robustness and good reproducibility of both methods. Conclusions: Both methods showed comparable performance, choice of method will rather be influenced by practical consideration.