Hydrophobic peptides are difficult to detect in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because of the hydrophilic properties of conventional matrices and the low affinity for hydrophobic peptides. Recently, we reported on alkylated dihydroxybenzoic acid (ADHB) as a matrix additive for hydrophobic peptides; however, the peptides were detected in the rim of the matrix-analyte dried spot. Here, we report on a novel matrix, alkylated trihydroxyacetophenone (ATHAP), which is a 2,4,6-trihydroxyacetophenone derivative incorporating a hydrophobic alkyl chain on the acetyl group and thus is expected to have an affinity for hydrophobic peptides. ATHAP increased the sensitivity of hydrophobic peptides 10-fold compared with α-cyano-4-hydroxycinnamic acid (CHCA), in which the detection of hydrophilic peptides was suppressed. The peptides were detected throughout the entire matrix-analyte dried spot using ATHAP, overcoming the difficulty of finding a "sweet spot" when using ADHB. In addition, ATHAP functioned alone as a matrix, unlike ADHB as an additive. In phosphorylase b digests analysis, hydrophobic peptides, which were not detected with CHCA for 1 pmol, were detected with this matrix, confirming that ATHAP led to increased sequence coverage and may extend the range of target analytes in MALDI-MS.