Background/purpose: This work was performed to optimize extraction conditions for D‐Squame® tape skin samples for use in the skin multiple analyte profile (SkinMAP) method, a Linco Research Corporation bead‐based assay for skin analytes. The experiments were designed to help identify sources of variability during extraction that may be amenable to further control.Methods: Two experimental designs were used to study factors influencing the extraction of skin samples from D‐Squame® tapes. Visually healthy skin samples were obtained from both female and male adult volar forearms. Factors studied in two experiments included: four surfactant (SDS) levels (0.02–0.2%), two buffer types [Citrate‐phosphate buffered saline at pH 5.5, phosphate‐buffered saline (PBS) at pH 7.4], two buffer volumes (1.0, 1.5 mL), two propylene glycol (PG) levels (0.1%, 1.0%), two extraction temperatures (7–10°C, 22–30+°C), two extraction times (30, 60 min), and location in sonication bath (two vectors). The response biomarkers were cortisol, fibronectin, human serum albumin, involucrin, keratin‐6 and keratins 1, 10. Skin sampling sites were also evaluated as sources of variation.Results: There was no single set of extraction conditions in our experiments that maximized recovery of all the biomarkers. SDS level had the most consistently significant (P<0.05) and directional effects on biomarker recoveries. In general, higher SDS resulted in higher recovery of all biomarkers. There was less consistency and fewer significant results for the other extraction factors.Conclusions: These data enable us to better manage SkinMAP studies and interpret their results. The use of 1.5 mL PBS containing 0.2% SDS and 0.5% PG with 30 min sonication at low (near 4°C) temperature is optimal for the quantitation of a range of SkinMAP analytes. In order to protect researchers from obtaining inflated false positive rates, it is crucial to design such studies and analyze the data using appropriate statistical methodology, especially for those studies involving only a small number of subjects.