Synthesis and biological evaluation of steroidal derivatives with anticancer properties is an active area of drug discovery. Here we measured the relative affinities of D-seco modified steroidal derivatives for estrogen receptor α, estrogen receptor β or androgen receptor ligand binding domains using an optimized non-transcriptional fluorescent cell assay in yeast. Ligand binding domains of steroid receptors were expressed in-frame with yellow fluorescent protein in the yeast Saccharomyces cerevisiae. Addition of known steroid ligands to yeast expressing the appropriate cognate receptor results in increased fluorescence intensity, enabling estimation of receptor binding affinities in a dose-response and time-dependent manner. Relative binding affinities of D-seco modified steroidal derivatives 1–4 were then evaluated using this yeast system by live cell fluorimetry and fluorescence microscopy, coupled with in vitro cytotoxicity and in silico molecular docking studies. D-Seco estratriene derivative 2displayed strong affinity for both estrogen receptor α and β ligand binding domains and negligible affinity for the androgen receptor ligand binding domain. Compound 2 also showed moderate cytotoxicity against estrogen receptor positive MCF-7 breast adenocarcinoma cells. In addition to identification of new ligands for steroid receptors, this assay could also be used to filter out compounds with potential for off-target interactions with steroid receptors during the early stages of compound screening.