Peak tailing of phosphate prodrugs in acidic mobile phases was thoroughly investigated. The results indicated that both metal-phosphate interactions and silanophilic interactions contributed to the observed peak tailing. Column pretreatment with phosphate buffers was demonstrated to be an effective and robust approach in suppressing metal-phosphate interaction. Silanophilic interactions, such as hydrogen bonding interactions between protonated isolated silanol groups and partially deprotonated phosphate groups were mobile phase pH dependent. The combination of column pretreatment and volatile low pH mobile phase buffers can be used to mitigate peak tailing issues in developing MS compatible RPLC methods for phosphate prodrugs. The use of non-endcapped columns should be avoided in RPLC analysis for phosphate prodrugs due to large amount of residual silanol groups in the stationary phases.