Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.