Vanda tricolor orchids are endemic plants that live on the slopes of Mount Merapi. The use of plant growth regulator for in vitro multiplication can affect the growth of orchid bud explants. This study aimed to determine the best combination of 2,4-D and TDZ in the growth of Vanda tricolor orchids. This research has been carried out in the In Vitro Laboratory of the Faculty of Agriculture, Universitas Muhammadiyah Yogyakarta in December 2018. This research was arranged in a single-factor Completely Randomized Design (CRD) consisting of 6 treatments namely 0 mg/ℓ 2,4-D + 0 mg/ℓ TDZ (control); 0 mg/ℓ 2,4-D + 0,5 mg/ℓ TDZ; 2 mg/ℓ 2,4-D + 0 mg/ℓ TDZ; 2 mg/ℓ 2,4-D + 0,5 mg/ℓ TDZ; 4 mg/ℓ 2,4-D + 0 mg/ℓ TDZ; 4 mg/ℓ 2,4-D + 0,5 mg/ℓ TDZ. Each treatment consisted of 3 replications, each replication consisted of 3 samples so that the total number was 54 units. The results showed that the medium, without 2,4-D and TDZ gave the best influence on the parameters of callus emergence 2 weeks, percentage of callus explants (22.22%), and pro-embryos emergence (2 weeks).