Govaniadine (1), a tetrahydroprotoberberine-type alkaloid, is one of the main active constituent isolated from Corydalis govaniana Wall., Papaveraceae, with several biological activities: antinociceptive, anti-urease, and leishmanicidal. Some articles reporting tetrahydroprotoberberine and structurally related alkaloids cytotoxicity prompted us to evaluate the influence of compound 1 on cellular viability in two different cell lines: human hepatoma carcinoma (HepG2) and human embryonic kidney (HEK-293T) and its permeation across the human colon carcinoma cell line (Caco-2). Cellular viability reduction of compound 1 was observed between 30 and 100 μM (HepG2) and between 70 and 100 μM (HEK-293T). However, the effects were weaker than those in the positive controls (T-2 toxin and camptothecin). Prior to proceed the transport studies, a method for compound 1 quantification in Hank’s Balanced Salt Solution medium was developed and validated by LC-MS/MS. The two calibration curves were linear over the concentration range of 6.3–200 nM and 0.2–10 μM. The apparent permeability coefficient for absorptive transport was 20.6 ± 3.9 × 10−6 cm/s, indicating compound 1 crossed the cell monolayer by passive diffusion and it was not subjected to active efflux. [Figure not available: see fulltext.] © 2020, Sociedade Brasileira de Farmacognosia.