Most molecular biology and biochemical analyses use cultured cells grown in anchorage-dependent monolayer conditions. The standard oncogenic transformation assay for cell lines is usually performed in soft agar rather than in monolayers because of the higher transformation efficiency of cells in soft agar. However, cells suspended in soft agar cannot be readily recovered for studying inducible biochemical and molecular events. We developed an over-agar assay that enables us to study tumor promoter-induced cell transformation and the associated biochemical or molecular events under anchorage-independent conditions.