The development of high-throughput single-cell RNA sequencing (scRNA-seq) has enabled access to information about gene expression in individual cells and insights into new biological areas. Although the interest in scRNA-seq has rapidly grown in recent years, the existing methods are plagued by many challenges when performing scRNA-seq on multiple samples. To simultaneously analyze multiple samples with scRNA-seq, we developed a universal sample barcoding method through transient transfection with short barcode oligonucleotides. By conducting a species-mixing experiment, we have validated the accuracy of our method and confirmed the ability to identify multiplets and negatives. Samples from a 48-plex drug treatment experiment were pooled and analyzed by a single run of Drop-Seq. This revealed unique transcriptome responses for each drug and target-specific gene expression signatures at the single-cell level. Our cost-effective method is widely applicable for the single-cell profiling of multiple experimental conditions, enabling the widespread adoption of scRNA-seq for various applications.