A protocol for the isolation and regeneration of protoplasts from leaf explant of Rhyncholaelia digbyana is presented. The protoplasts were isolated using hemicellulase enzymes at 1.5, 2.25, and 3% (w/v), pectinase at 0.5 and 0.75% (w/v) and cellulase at 1 and 2% (w/v). Protoplast counting was carried out with a Neubauer camera and an optical microscope at 40X, and viability was determined with Evans blue dye at 0.025% (w/v). The protoplasts were cultivated following the standard plate method, using the K&M media with 0.06 M of saccharose, 2.3% of Gelrite and plant growth regulators. It produced a yield of 386250±1875 protoplasts/g of tissue using an enzymatic combination of 1.5% (w/v) of hemicellulase, 0.5% (w/v) of pectinase and 1% (w/v) of cellulase with an incubation time of 4 h. The colonies were observed after two months of culture and the highest number of colonies (1.66±0.50) was obtained when the protoplasts were cultured in Kao medium with 4.53 µM of 2,4-Dichlorophenoxyacetic acid (2,4-D), 0.912 µM of Zeatin and 3.10 µM of 6-Benzylaminopurine (BA). The nuclear DNA content estimated by flow cytometry for R. digbyana was 27.39±3.8 pg of DNA equivalent to 26.79×109 pb and the number of mitotic chromosomes counted was 2n=40. Key words: Isolation, in vitro culture, plant regeneration, flow cytometry.