We developed a simple method for determining the relative amount of α-smooth muscle actin (αSMA) produced in fibroblasts. The principle of the method is based on an enzyme immunoassay (EIA) for αSMA in microcultured fibroblasts. The optimized protocol of the assay is as follows. Human fibroblasts were cultured with transforming growth factor β1 (TGFβ1) in a microtiter plate and directly immobilized on the plate. The αSMA produced was labeled and subjected to indirect enzyme immunoassay using alkaline phosphatase, and optical density was measured. Semiquantitativeness was confirmed using various numbers of cells in which αSMA production was induced by treatment with TGFβ1. The assay simply demonstrated that interferon-γ (INF-γ) inhibited the production of αSMA in an established cell line and that in primary cultured cells originated from the contractile nodule. Since the assay is simple and semi-quantitative, it is useful for elucidating the mechanism of contractile diseases and screening a large number of substances that have an inhibitory effect on the change in activity of myofibroblasts.