The aim of this study was to develop an efficient protocol for shoot tip culture from adult plants of Paphiopedilum Pfitzer. A considerable seasonal effect on explant collection was observed in the aseptic cultures established from adult plants, including the survival and microbial contamination of explants. The shoot tip explants excised from adult plants in February and May showed higher survival and had less contamination than those explants excised in August and November. Moreover, the season of explant collection also affected the subsequent shoot forming capacity and multiplication of axillary buds. In Paphiopedilum ‘In-Charm Silver Bell’, higher shoot forming capacity was observed in February and May, whereas higher shoot multiplication was observed only in February. In Paphiopedilum ‘Hsinying Maudiae Leopard’, both February and May were optimal timing for shoot forming capacity and multiplication. We also demonstrated the effectiveness of transcinnamic acid (tCA), an antiauxin chemical in diminishing the apical dominance of shoot tip explant and thus improving the axillary bud outgrowth. In P. ‘In-Charm Silver Bell’, the addition of 100 μM tCA plus 13.3 μM 6-benzylaminopurine (BA) for 1 month promoted axillary shoot bud formation from shoot tip explants as compared with the control.