Phytochemical investigation of the dichloromethane extract of the dried aerial parts of Corydalis rupestris (Papaveraceae) resulted in the identification of four new isoquinoline alkaloids rupestrines A-D and one known isoquinoline alkaloid, namely, stylopine. The structures of these compounds were characterized by extensive spectroscopic methods including 1D- ((1)H and (13)C) and 2D NMR experiments (COSY, HSQC, HMBC, and NOESY) as well as HRESIMS analyses. In addition, the absolute configurations of rupestrines A-D were determined using modified Mosher's method. Cytotoxic effects of alkaloids and their interaction with albumin were also investigated in this study. CI - Copyright (c) 2018 Elsevier Inc. All rights reserved.