A spectrophotometric assay based on the color reaction between N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate (DMPAP) and 4-[4-(dipropylamino) phenylazo]-benzenesulfonic acid (propyl orange) was used to determine DMPAP concentrations as a wood preservative. The assay was carried out using a propyl orange solution at pH 2.9. The visible absorption spectrum of propyl orange showed an absorption maximum at 510 nm, which decreased linearly with increasing DMPAP concentration from 0 to 10 ppm. To apply this assay method to determine DMPAP retention in treated wood, the influence of wood extractives on the assay was investigated. Extractives from Japanese cedar, hinoki cypress, and Japanese larch were found to increase apparent DMPAP concentration. However, it was also found that measuring visible absorption at 477 nm prevented overestimation and gave precise values. This assay can be a viable alternative to the current methods for the determination of DMPAP concentrations.