X chromosome activity is a defining attribute of naive pluripotency, with naive pluripotency being a rare context in which both X chromosomes of females are active. RNA-fluorescence in situ hybridization (RNA-FISH) is a powerful tool to determine the transcriptional status of specific genes with allelic and single-cell resolution and has been widely used in the context of X chromosome inactivation, the process ensuring dosage compensation for X-linked genes between sexes in mammals. RNA-FISH using genomic or intronic probes allows the detection of newly synthesized transcripts at the site of transcription. This technique is invaluable for appreciating the putative heterogeneity in the expression profiles within cell populations. RNA-FISH has the added advantage of allowing the visualization of gene transcription in a spatial perspective. Here, we provide a detailed protocol describing the application of RNA-FISH to detect nascent X-linked transcripts in female naive human embryonic stem cells to assess their X chromosome status, along with another complementary technique, DNA-FISH.