There is a large body of evidence supporting the beneficial role of adipose-derived stem cells (ASCs) in tissue repair and regeneration. These effects appear to be mainly mediated by paracrine signaling pathways and enhanced during hypoxia. Mass spectrometry (MS) represents a valuable tool for proteomic profiling of cultured ASCs, which may help revealing the identity of the factors secreted by the cells under different conditions. However, serum starvation essentially required to obtain samples compatible with secretome analysis by MS can have significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effect of hypoxia on the ASC proteomic profile.