The cytochrome P450 enzyme, CYP27a1, converts cholesterol into 27-hydroxycholesterol, and both the metabolite and the enzyme are associated with breast cancer, promoting proliferation and metastasis. CYP27A1 is enriched in cancer tissues, and the amount of CYP27a1 correlates to tumor grade. Hence, CYP27a1 is a potential biomarker, and a method for determination and quantification is needed. Antibody assays shows limited performance, and therefore the goal of this work was to develop a nano liquid chromatography mass spectrometry (LC-MS) method for determination of CYP27a1. A sample preparation workflow for CYP27a1 in cell samples was investigated. CYP27a1 was identified by nano LC-MS in MDA-MB-231 breast cancer cells by fractionating cell lysates using gel electrophoresis. Enrichment of CYP27a1 in cell samples using immunoprecipitation, an antibody based method for protein extraction, did not provide adequate identification. However the former method works when looking at other proteins, and this confirms that there are limitations in using antibody based methods for determination and quantification of CYP27a1.