To clarify the relationships of the commonly used spectral measurements and the in vivo activity of b-carboline intercalators N-[2(3-carboxyl-9-benzyl-carboline-1-yl)ethyl-1-yl]-aspartic acid (3a), -tyrosine (3b), -serine (3c), and -threonine (3d) were prepared as the model compounds by using a three-step procedure. In UV measurements of calf thymus DNA (CT DNA) the in vivo active 3a–c induced both hypochromic effect and hypsochromic shift, while the in vivo inactive 3d did not. In CD measurements the in vivo active 3a–c changed the intensities of both positive and negative bands of CT DNA in same direction, while the in vivo inactive 3d induced an opposite change. In fluorescence measurements due to adding CT DNA the increase of the fluorescence intensities of 3a–c were 10–31% higher than that of 3d, and the bathochromic shifts of 3a–c were 2–6 nm higher than that of 3d. The viscositic experiments revealed that 3d possessed the lowest binding ratio. The melting curves indicate that the effect of 3a–c (DTm, 10.8–12.9C) on the Tm of CT DNA was significantly higher than that of 3d (DTm, 4.9C). Spectroscopic, viscositic, and melting curve investigations are not only able to provide multiple evidences for DNA intercalation mechanism, but also are able to distinguish the in vivo active and inactive N-[2(3-carboxyl-9-benzyl-carboline-1-yl)-ethyl-1 yl]-amino acids.