We characterized the bis-quinolizidine tetracyclic alkaloid (5S, 6S, 7R, 11R)-matrine in a supersonic jet expansion, using chirped-pulsed broadband microwave spectroscopy. Previous crystal diffraction analyses suggested 16 diastereoisomers associated with matrine's four carbon stereocenters but were inconclusive whether the lactamic nitrogen atom would additionally produce separated trans-/cis- diastereoisomers or if both species may interconvert through low potential barriers. Our experiment simultaneously detected trans- and cis-matrine through their rotational spectrum, confirming the possibility of conformational rearrangement in matrine alkaloids. The two matrine conformers mainly differ in the envelope or half-chair lactamic ring, as evidenced by the experimental rotational and nuclear quadrupole coupling parameters. Molecular orbital calculations with ab initio (MP2) and density functional methods (B3LYP-D3(BJ) and MN15) were tested against the experiment, additionally offering an estimation of the cis-/trans- barrier of 24.9-26.9 kJ mol-1. The experiment illustrates the structural potential of chirped-pulsed broadband microwave spectroscopy for high-resolution rotational studies of biomolecules in the range of 20-40 atoms.