Summary Background Penetration, autoxidation and N‐acetylation of p‐phenylenediamine (PPD) have been studied in vitro and ex vivo. However, a clear understanding of in vivo PPD penetration and the formation of PPD derivatives is lacking. Objectives To obtain insights into the in vivo penetration, clearance and formation of PPD derivatives in human skin. Methods Patch test chambers containing PPD 1% pet. were applied to the forearms of two human volunteers, with increasing application times. Non‐invasive Raman microspectroscopy was used for detection of PPD (derivatives) in skin at several follow‐up times. Results Application of a PPD 1% pet. patch for 30 min resulted in substantial amounts of PPD in the stratum corneum of 90 mg PPD/g keratin. PPD contents were highest after three applications for 1 h each (330 mg PPD/g keratin), followed by single applications for 2 h 40 min, 2 h, and 23 h. The PPD half‐time in the skin was 3 h. No spectral contributions of Bandrowski's base, monoacetyl‐PPD and diacetyl‐PPD were detected. Conclusions We have gained insights into the in vivo penetration of PPD in human skin by using non‐invasive Raman spectroscopy. Penetration into the skin was fast, and the PPD concentrations detected in the stratum corneum were high. PPD was detected in both the stratum corneum and the viable epidermis. Oxidized or acetylated PPD derivatives could not be detected.